Protection against environmental toxicity through manipulation of the processing of messenger RNA precursors

ABSTRACT

This invention describes the identification of pre-messenger RNA processing as a novel target of environmental stress caused for example by lithium and sodium toxicity. Overexpression of different types of proteins (or protein fragments) from different organisms but all involved in pre-mRNA processing, protects yeast from salt stress, which indicates that any stimulation of this process, independently of its mechanism, may counteract the toxic effects of mineral salts. A similar phenotype of tolerance to NaCl and to LiCl has been observed by overexpression of these types of proteins in transgenic  Arabidopsis  plants, demonstrating the generality of this protective effect in eukaryotic cells and organisms.

FIELD OF THE INVENTION

This invention refers to the use of nucleic acids and proteins involvedin the processing of messenger RNA precursors for the enhancement of oftolerance to environmental stress such as mineral salt toxicity ineukaryotic cells and organisms.

BACKGROUND TO THE INVENTION

The nature of the cellular targets sensitive to lithium and sodiumtoxicity represents an important gap in our knowledge on the physiologyof ion homeostasis in eukaryoUc cells. The characterisation of thesetargets is essential for the understanding of clinical problems such asthe effects of lithium on the therapy for dipolar disorder [Schou (1997)Arch, Gen. Psychiatry 54, 9] or high sodium levels associated withhypertension [Lifton (1996) Science 272, 676). Another problem,completely different but also related to ionic homeostasis, is theprogressive salinisation of cultivated lands subjected to intensiveirrigation, which has turned crop plant breeding for salt tolerance intoan urgent need for the development of a sustainable agriculture in aridregions [Serrano (1996) Int. Rev. Cytol. 165; 1; Yeo (1998) J. Exp. Bot.49, 915; Holmberg & Bülow (1998) Trends Plant Sci. 3, 61].

Apart from ion transport [Haro et al. (1991) FEBS Lett. 291, 189;Gaxiola et al. (1999) Proc. natl. Acad. Sci. USA 96, 1480; Apse et al.(1999) Science 285, 1256] and osmolyte synthesis [Tarczynski et al(1993) Science 259, 508; Kishor et al. (1995) Plant Physiol. 108, 1387:Alia et al. (1998) Plant J. 16, 155], the manipulation of cellularsystems most sensitive to high ion concentrations and to water stressoffers alternative routes to improve salt tolerance of crop plants[Serrano (1996) Int. Rev. Cytol. 165, 1; Tezara et al. (1999) Nature401, 914].

Genetic and biochemical analyses have allowed to identify the product ofthe yeast gene HAL2 as an important physiological target of salttoxicity [Gläser et al. (1993) EMBO J. 12, 3105; Dichtl et al. (1997)EMBO J. 16, 7184]. HAL2 encodes a 3′,5′-biphosphate nucleotidase, whichis very sensitive to inhibition by lithium and sodium [Murguía et al.(1995) Science 267, 232]. Salt inhibition of Hal2p results in theintracellular accumulation of 3′-phosphoadenosine 5′-phosphate (pAp)[Murguía et al. (1996) J. Biol. Chem. 271, 29029], a toxic compoundwhich in turn inhibits the reactions of reduction and transfer ofsulphate groups, as well as some exoribonucleases [Dichtl et al. (1997)EMBO J. 16, 7184; Gil-Mascarell et al. (1999) Plant J. 17, 373]. Thereare genes homologous to HAL2 in plants [Gil-Mascarell et al. (1999)Plant J. 17, 373] and in mammals [López-Coronado et al. (1999) J. Biol.Chem. 274, 16043] although in the latter case the encoded enzyme isinhibited by lithium but not by sodium.

The salt tolerance conferred by overexpression of Hal2p, the wild-typeprotein as well as mutated versions resistant to lithium and sodium, isrelatively modest [Albert et al. (2000) J. Mol. Biol. 295, 927]. Thissuggests the existence of additional targets of salt toxicity, whichbecome limiting once the HAL2 bottleneck is overcome, but the nature ofthese important salt-sensitive processes is not yet known.

Two patent applications relating to osmotic stress but describingprotective mechanisms different to the general mechanism described inthe present invention are the following: ES2110918A (1998-02-16)relating to the production of plants tolerant to osmotic stress throughthe manipulation of carbohydrate metabolism and ES2134155A1 (1998-10-07)relating to a method to confer tolerance to osmotic, water and saltstress in glycophylic plants, through the use of genes encoding proteinswith peroxidase activity.

SUMMARY OF THE INVENTION

The technical problem underlying the present invention is to provide amethod that can be used to enhance stress tolerance of cells andorganisms that suffer from stress conditions like osmotic stress, causedby salt, drought or cold and freezing stress.

A solution to this technical problem is achieved by providing protectionagainst osmotic stress in cells and organisms through the manipulationof the processing of messenger RNA. Provided by the present invention isat set of isolated genes that are able to confer to a heterologeous hostcell or host organism tolerance to stress conditions. These genes areall involved in the processing of mRNA precursors (such as synthesis,splicing, 5′ and 3′ end modification, movement, transport to thecytoplasm, metabolism etc. . . ) and they all showed a salt resistancephenotype when separately transformed to a salt sensitive yeast mutant.By doing so each gene acted as an efficient enhancer of stresstolerance, without the assistance of additional factors.

This set of genes, comprising SR-like proteins, (nuclear) RNA bindingfactors, components of ribonucleoprotein complexes, transcriptionfactors, and nuclear movement proteins, enables the person skilled inthe art to genetically alter the organism of interest in order to makeit tolerant to stress situations such as osmotic stress situations, moreparticularly mineral salt or Na+ or Li+ toxicity. Each of the disclosedgenes enables the person skilled in the art to modify cell fate and/orplant development and/or biochemistry and/or physiology by introducingat least one of these genes into the cell. For the cultivation of cropplants for example, of which many are sensitive to stress conditionslike salt, drought or cold, the disclosed genes offer the possibility tosolve the problem of reduced yield and reduced economic profit.

This invention offers a solution to cellular toxicity caused byenvironmental stress through the manipulation of the processing ofmessenger RNA precursors and the embodiments of the invention comprisemethods, nucleic acids, polypeptides, vectors, host cells and transgenicorganisms like transgenic plants.

DETAILED DESCRIPTION OF THE INVENTION

Soil salinity is one of the most significant abiotic or environmentalstresses for plant agriculture. Apart from the practical goal ofgenetically improving the salt tolerance of crop plants, salt toleranceresearch represents an important part of basic plant biology. Alsoresearch on two other major abiotic stresses, drought and cold, isintimately linked with salt stress work. For example, many genes thatare regulated by salt stress are also responsive to drought or coldstress (Zhu J. K., 1997, Molecular aspects of osmotic stress in plants,CRC Crit. Rev. Plant Sci. 16 253-277). A person skilled in the art thuscan assume that when an isolated gene confers salt tolerance to a hostorganism when transfected herein, it could also confer cold and/ordrought stress tolerance. Salt, drought and cold are considered to bethe three most important forms of osmotic stress.

In order to identify novel targets of environmental toxicity ineukaryotic cells, the inventors characterised genes conferring anincrease in the tolerance to mineral salts when expressed in yeastcells. Therefore, cDNA libraries from Arabidopsis thaliana [Minet et al.(1992) Plant J. 2, 417] and Beta vulgaris were searched which couldconfer tolerance to salt stress by overexpression in yeast cells, inwhich accumulation of pAp, and its toxic effect, was avoided by additionof methionin to the culture medium[Gläser et al. (1993) EMBO J. 12,3105; Dichtl et al. (1997) EMB J. 16, 7184; Murguía et al. (1995)Science 267, 232; Murguía et al. (1996) J. Biol. Chem. 271, 29029]. Theresult of this strategy has given rise to the present invention. This isa functional approach to identify genes and proteins that are involvedin the response of plants to salt stress. For this purpose cDNAexpression libraries were constructed as described in example 1 andexample 3 and competent yeast strains (see example 2) were used toscreen cDNAs that increased the yeast salt tolerance uponoverexpression. The growth of these yeast strains is normally inhibitedat high NaCl or LiCl concentrations of (150 mM) similar to thoseimpairing growth of most crop species. After transforming the yeastcells with the cDNA library, colonies were pooled and selected for theirability to grow in the presence of 150 mM NaC1. This screening procedureis further described in example 4.

Isolation of Arabidopsis thaliana Genes that Enhance Salt Tolerance

A cDNA library from the plant Arabidopsis thaliana of ca 7,5×10⁵transformants, was screened and three independent clones were isolatedwhich were able to confer tolerance to high salt concentrations in yeast(FIG. 1). Surprisingly, these three cDNAs encode proteins implicated inthe processing of messenger RNA precursors. These three proteins werenamed Ct-SRL1, RCY1 and U1A.

Two of these proteins belong to the family of the so-called “SR-like” or“alternating arginin-rich” factors, defined by having a domain with ahigh content in Arg residues alternating with Ser, Asp, andlor Glu (RSdomain) (FIG. 2). Members of this family have been involved inconstitutive and/or alternative splicing, and in the coupling ofdifferent steps during processing and metabolism of messenger RNA(transcription, modifications at the 5′ and 3′ ends, and pro-mRNAsplicing, transport of mature RNA to the cytoplasm, etc.). The Ct-SRL1protein, with amino acid composition as set forth in SEQ ID NO. 3, isencoded by the cDNA identified herein as SEQ ID NO.1, which can also befound on the genomic region of Arabidopsis thaliana chromosome 5, P1clone MNJ8 (Genbank accession number AB017069). Also the proteinsequence of SEQ ID. NO. 3 is present in the public database under theaccession number BABO9109 but no function was assigned to thissequence.The cDNA of Ct-SRL1 is not full-length and it encodes thecarboxy-terminal end of a putative SR-like protein, which includes theRS domain. Its expression in yeast confers tolerance to lithium andsodium (FIG. 1).

The second clone encodes a putative protein with an N-terminal cyclindomain related to those of K-type and T-type cyclins and with anarginin-rich domain at the C-terminus (FIG. 2 A). This SR-like proteinwas named RCY1 for altering arginin-rich cyclin 1. Its expression inyeast conferred tolerance to lithium and sodium (FIG. 1) To determinethe role of the RS-domain in this SR-like protein for the enhancement ofsalt tolerance in yeast, the cyclin domain and the RS domain of RCY1were separately cloned and transformed into yeast cells. The division ofboth domains is marked in FIG. 2A by the underlined methionin residueand the C-terminal part, containing the RS domain is referred to as SEQID NO. 21 (FIG. 5). The expression of only the RS domain confers thesame phenotype as expression of the full-length protein (FIG. 3). Theseresults demonstrate that, in the case of “SR-like” factors, it is theexpression of the RS domain per se, and not of a specific protein, whichconfer salt stress tolerance. The amino acid composition of RCY1 proteinis represented herein as SEQ ID NO. 4. The cDNA of RCY1 is identifiedherein as SEQ ID NO. 2 and this sequence can be found on the genomicregion of Arabidopsis thaliana chromosome II sequence from clone T9J22.The nucleotide sequences of this genomic Arabidopsis thaliana clone,that corresponds to a part of the nucleotide sequence of the RCY1 gene,is annotated as a putative cyclin (accession number AAC14513.1). Thisprotein prediction from the public database, lacks the first 55 aminoacids of the RCY1 protein. As such, the fragment of the RCY1 proteincorresponding with the amino-terminal region is represented in SEQ ID NO22.

The third Arabidopsis clone encodes a U1A protein from Arabidopsis, acomponent of the U1-snRNP, the ribonucleoprotein complex whichrecognises the 5′-splice site in one of the first steps in theprocessing of introns of the pre-messenger RNA. The U1A protein ofArabidopsis is previously described by Simpson et al (1995, EMBO J. 14:4540-4550) as a specific component of the spliceosomal U1-snRNP. Theexpression of U1A in yeast conferred weaker LiCl tolerance and notolerance to NaCl (FIG. 1). The U1A protein, with amino acid compositionas set forth in SEQ ID NO 6, is encoded by the cDNA identified herein asSEQ ID. NO. 5. The protein sequence as well as the nucleic acid sequencecan be found in the public database under the accession numbersCAA90283.1 and Z49991 respectively and are fully annotated. Thesequences described above are presented in FIG. 5.

The phenotypes as described above were observed in the presence andabsence of methionine, and in different genetic backgrounds.

The improvement of salt tolerance by expression of the Arabidopsisclones was not due to the stimulation of ion transport in yeast, sinceit was not associated to changes in the intracellular lithiumconcentrations. This was determined as described in example 5. Also thephenotypes were maintained in a yeast strain defective in vacuolartransport.

The above-mentioned data suggested that an impact on another cellularprocess was responsible for the observed stress tolerance. The inventorsbelieved that processing of messenger RNA precursors could be a target,of ionic toxicity in eukaryotic cells. This has not been describedpreviously.

In agreement herewith, the inventors have confirmed that processing ofintrons of pre-mRNAs is inhibited in yeast in the presence of, forexample, lithium chloride. Two independent tests for pre-mRNA splicingin vivo supported this invention. First, the inventors have measured thespecific activity of the enzyme β-galactosidase synthesised in yeastcells from a plasmid containing the E. coli LacZ gene artificiallyinterrupted by an intron (example 6). They detected a decrease in theaccumulation of this enzyme, as compared to that produced from thecontrol construct without intron, when LiCl is added to the culturemedium. Simultaneous expression of the RS domain of Arabidopsis SRL1 inthese yeast cells partially blocked the observed inhibition (data notshown). These results have been confirmed by the second assay, in whichthe inventors determined directly, by the RT-PCR technique as describedin example 7, the inhibition of splicing in the presence of lithium. Theaccumulation of endogenous yeast messenger RNA precursors in thepresence of LiCl, for example the pre-mRNA corresponding to the SAR1gene, was demonstrated. Because a general inhibition of splicing wouldfirst affect the removal of those introns normally processed with lowerefficiency, the inventors choose for these experiments the SAR1pre-mRNA, which contains such an intron (Kao and Siliciano, 1996, Mol.Cell. Biol. 16: 960-967). Here again, the inventors observed theaccumulation of SAR1 pre-mRNA by incubating yeast cells under saltstress conditions, and how it was partially reversed by simultaneousco-expression of Ct-SRL1. In this way the inventors demonstrated thatthe inhibition of processing precursor mRNA in the presence of salt ispartially reverted by expression of one of the Arabidopsis clonesmentioned before (e.g. Ct-SRL1).

The general significance of the present invention was corroborated bythe phenotype of transgenic Arabidopsis plants, which overexpressed theCt-SRL1 cDNA. Supporting the general character of the mechanism, theexpression of the same Ct-SRL1 cDNA in under control of the CaMV 35Spromoter Arabidopsis transgenic plants, increases their tolerance toNACl and LiCl in a similar way as in yeast. This can be observed, forexample, by germination of transgenic seeds in agar plates containingLiCl concentrations which are toxic to wild-type control seeds (FIG. 4).The three independent transgenic lines were able to grow indicating theefficiency of the method of the present invention.

From these results and from the very nature of the isolated Arabidopsisclones, it can be deduced that any stimulation of the processing ofmessenger RNA precursors, independently of the mechanism involved,counteracts the toxic effect of the salt, and that this protectiveeffect against salt stress is general in all eukaryotic cells andorganisms.

The universal character of the invention, namely that the protectiveeffect against salt stress is not species-dependent, was confirmed bythe isolation of sugar beet genes and eukaryotic genes which also confersalt tolerance and which were also related to the processing ofmessenger RNA precursors.

Isolation of Beta Vulgaris Genes that Enhance Salt Tolerance

Another aspect of the present invention is the procedure of screening acDNA library from NaCl induced sugar beet leaves and subsequentisolation of the seven sugar beet genes that confer stress tolerance toyeast cells. A functional approach to identify sugar beet genes andproteins that are involved in the response of plants to salt stress wasfollowed. For this purpose a NaCl-induced cDNA expression library wasconstructed from sugar beet leaves as described in example 1 and example3 and the Na⁺-sensitive yeast mutant strain JM26 (see example 2) wasused to screen for sugar beet cDNAs that increased the yeast salttolerance upon overexpression. The growth of this yeast mutant isnormally inhibited at NaCl concentrations (150 mM) similar to thoseimpairing growth of most crop species. After transforming the yeastcells with the cDNA library, colonies were pooled and selected for theirability to grow in the presence of 150 mM NaCl. This screening procedureis further described in example 4. Six positive clones which survivedthe high concentrations of salt, were further characterised andcontained a gene with a sequence as in SEQ ID NO.7, SEQ ID NO 9, SEQ IDNO 11, SEQ ID NO. 13, SEQ ID NO. 15 or SEQ ID NO. 17. The correspondingprotein sequences encoded by these genes have an amino acid compositionas set forth in SEQ ID NO 8, SEQ ID NO. 10, SEQ ID NO 12, SEQ ID NO. 14,SEQ ID NO. 16 or SEQ ID NO. 18 respectively. All these sequences arepresented in FIG. 5. Surprisingly, each of the six selected yeast clonesare transformed by a gene that encodes a protein which is implicated inthe processing of mRNA: one protein is a putative arginine-aspartate RNAbinding protein, two are a putative RNA binding protein, two are aputative transcription factor and one shows similarity to a nuclearmovement protein. Therefore these proteins (and the encoding genes) aresuitable to be used as stress tolerance enhancers through themanipulation of pre-mRNA processing, as is described for the Arabidopsisthaliana genes of the present invention and as described above.

The selected genes and their encoded proteins are further described inthe following paragraphs.

Accordingly, the invention relates to a novel isolated nucleic acid ofred beet as set forth in SEQ ID NO. 7, encoding a putativearginine-aspartate RNA binding protein and capable of enhancing salttolerance in yeast cells. The open reading frame, starting at nucleotideposition 51 and ending at position 1079 encodes the amino acid sequenceas set forth in SEQ ID NO. 8. This polypeptide has 76% identity and 86%similarity with the Arabidopsis thaliana putative arginin-aspartate RNAbinding protein (Swiss prot accession number AAB68037). The amino acidcomparison was done with the program GAP (Symbol comparison table:blosum62. cmp, CompCheck: 6430, BLOSUM62 amino acid substitution matrix,Reference: Henikoff, S. and Henikoff, J. G. (1992). Amino acidsubstitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA89: 10915-10919.). This program was also used for comparing the aminoacid sequences of the following red beet polypeptides.

The invention also relates to a novel isolated nucleic acid of red beetas set forth in SEQ ID NO. 9, encoding a putative RNA binding proteinand capable of enhancing salt tolerance in yeast cells. The open readingframe, starting at nucleotide position 14 and ending at position 625encodes the amino acid sequence as set forth in SEQ ID NO. 10. Thispolypeptide has 68% identity and 78% similarity with the Arabidopsisthaliana putative RNA binding protein (Swiss prot accession numberAAG52616.1).

The invention also relates to a novel isolated nucleic acid of red beetas set forth in SEQ ID NO. 11, further referred to as clone or sequencenumber 10, encoding a putative transcription factor and capable ofenhancing salt tolerance in yeast cells. The open reading frame,starting at nucleotide position 51 and ending at position 1119 encodesthe amino acid sequence as set forth in SEQ ID NO. 12. This polypeptidehas 81% identity and 86% similarity with the Spinacia oleracea nuclearRNA binding protein (Swiss prot accession number AAF14145.1).

Nucleotides 1 to 475 from SEQ ID NO. 11 were published in the ESTdatabase under the accession number BF011019 with the description of aDNA fragments of a Sugar beet germination cDNA library and that issimilar to a putative transcription factor.

The invention also relates to a novel isolated nucleic acid of red beetas set forth in SEQ ID NO. 13, encoding a putative transcription factorand capable of enhancing salt tolerance in yeast cells. The open readingframe, starting at nucleotide position 51 and ending at position 1121encodes the amino acid sequence as set forth in SEQ ID NO. 14. Thispolypeptide has 81% identity and 87% similarity with the Spinaciaoleracea nuclear RNA binding protein (Swiss prot accession numberAAF14144.1).

The invention also relates to a novel isolated nucleic acid of red beetas set forth in SEQ ID NO. 15, encoding a putative RNA binding proteinand capable of enhancing salt tolerance in yeast cells. The open readingframe, starting at nucleotide position 2 and ending at position 970encodes the amino acid sequence as set forth in SEQ ID NO. 16. Thispolypeptide has 68% identity and 74% similarity with the Oryza sativaputative RNA binding protein (Swiss prot accession number AAG59664.1).

The invention also relates to a novel isolated nucleic acid of red beetas set forth in SEQ ID NO. 17, encoding an unknown type of protein andcapable of enhancing salt tolerance in yeast cells. The open readingframe, starting at nucleotide position 35 and ending at position 922encodes the amino acid sequence as set forth in SEQ ID NO. 18. Thispolypeptide has 61% identity and 69% similarity with the Arabidopsisthaliana protein with similarity to a nuclear movement protein (Swissprot accession number BAA97317.1).

Accordingly, a preferred embodiment of the present invention relates toa method for induction of stress tolerance to an organism comprising theexpression of a (or at least one) Beta vulgaris gene, which is involvedin the processing of messenger RNA precursors.

Also the screening and selection procedure as described above can beused to select genes from other organisms than plants. As an example, asequence of Mus musculus was selected that enhances salt tolerance inyeast cells. This sequences is set forth in SEQ ID NO. 19 and it encodesa putative small subunit of an U2 snRNP auxiliary factor protein and iscapable of enhancing salt tolerance in yeast cells. The open readingframe, starting at nucleotide position 37 and ending at position 969encodes the amino acid sequence as set forth in SEQ ID NO. 20. Thispolypeptide has 78% identity and 84% similarity with the Arabidopsisthaliana U2 snRNP auxiliary factor, small subunit (Swiss prot accessionnumber BAB10638.1). This sequence is presented in FIG. 5.

This result illustrates that mammalian genes can also be use in themethod of the present invention. The method of the present invention isthus generally applicable to confer stress tolerance to a host cell ororganism through the manipulation of messenger RNA precursors.

The surprisingly strong phenotype of some of the yeast clones selectedas described above and the fact that these genes in an isolated positionand in a heterologous background acted as stress tolerance enhancers,makes these genes very attractive tools to induce stress tolerance inany organism of interest, without the need for accessory compounds. Theability of these genes to enhance osmotic stress tolerance, particularlymineral salt stress such as Na+ and Li+ stress in yeast cells whenisolated and transfected herein, clearly demonstrates their potential toconfer on their own osmotic stress tolerance to any heterologeous hostorganism. Alternatively, each of the stress tolerance genes of thepresent invention can be combined with another gene, in order to altercell fate or plant morphology, plant development, plant biochemistry orplant physiology.

According to a first embodiment the present invention relates to amethod to enhance stress tolerance in cells and organisms comprising themanipulation of the process of processing messenger RNA precursors.

According to a preferred embodiment, said stress could be environmentalstress, such as but not limited to osmotic stress, salt stress, droughtstress, cold or freezing stress.

According to a preferred embodiment, the methods of the invention relateto the enhancement of salt tolerance of cells and organisms.

Another embodiment of the invention relates to a method to protect cellsand organisms against salt toxicity comprising the manipulation of theprocess of processing messenger RNA (mRNA) precursors.

According to one embodiment, one way of manipulating the process ofprocessing messenger RNA precursors is by genetic or biochemicalmanipulation of at least one molecule which is involved in or whichinterferes with the process of processing messenger RNA precursors orwith one of the pathways of processing mRNA precursors.

The term “cells” relates to any prokaryotic or eukaryotic cell. The term“organism” relates to any mono- or multicellular organism of prokaryoticor eukaryotic origin.

The present invention clearly describes several genes and proteinsbelonging to different classes of genes and proteins which can be usedto enhance stress tolerance. These genes and proteins of the inventionhave been shown to have an effect on the process of mRNA processing.

Therefore, according to yet preferred embodiments, the invention relatesto any of the above-mentioned methods wherein the genetic or biochemicalmanipulation of a protein possessing a domain with a high content inArg-Ser, Arg-Glu and Arg-Asp dipeptides (RS domain), an RNA bindingprotein, a component of the U1-snRNP or the U2-snRNP complex, atranscription factor, or a nuclear movement protein is involved.

The invention also relates to the use of an isolated nucleic acidcomprising a nucleic acid sequence as represented in SEQ ID NO 1 with anamino acid sequence as set forth in SEQ ID NO 3, or a nucleic acid asrepresented in SEQ ID NO 2 with an amino acid sequence as set forth inSEQ ID NO 4 or SEQ ID NO 21, or a nucleic acid encoding a polypeptidecomprising the amino acid sequence represented in SEQ ID NO 22, for atleast one, or in at least one, of the above described methods. SEQ IDNOs 1 and 2 share substantial homology with genes encoding SR-likeproteins.

The invention also relates to an isolated nucleic acid comprising anucleic acid sequence as represented in SEQ ID NO 5 with an amino acidsequence as set forth in SEQ ID NO 6 for any of the above describedmethods. SEQ ID No 5 shares substantial homology with genes encoding acomponent of the I1-snRNP or the U2-snRP complex.

The invention also relates to an isolated nucleic acid comprising anucleic acid sequence as represented in any of SEQ ID NOs 7, 9, 11, 13,15, 17 or 19, with an amino acid sequence as set forth in any of SEQ IDNO 8, 10, 12, 14, 16, 18 or 20 for any of the above-described methods.SEQ ID NOs 7, 9, 13 and 15 share substantial homology with genesencoding RNA-binding proteins. SEQ ID NO 19 shares substantial homologywith genes encoding a component of the I1-snRNP or the U2-snRP complex.SEQ ID NOs 11 and 17 share substantial homology with genes encodingtranscriptional factors.

The invention further relates to an isolated nucleic acid encoding aprotein or an immunologically active and/or functional fragment of sucha protein selected from the group consisting of:

-   -   a) a nucleic acid comprising a DNA sequence as given in any of        SEQ ID NOs 1, 2, 5, 7, 9, 11, 13, 15, 17 or 19 or the complement        thereof,    -   b) Nucleic acid comprising the RNA sequence corresponding to any        of SEQ ID NOs 1, 2, 5, 7, 9, 11, 13, 15, 17 or 19 as in (a) or        the complement thereof,    -   c) Nucleic acid specifically hybridizing tot the nucleotide        sequence ad defined in (a) or (b),    -   d) nucleic acid encoding a polypeptide or protein with an amino        acid sequence which is at least 50%, preferably at least 60%,        70% or 80%, more preferably at least 85% or 90%, most preferably        95% identical to the polypeptide represented in any of SEQ ID        NOs 3, 4, 6, 8, 10, 12, 14, 16, 18, 20or 21,    -   e) nucleic acid encoding a polypeptide or protein comprising the        amino acid sequence as given in any of SEQ ID NOs, 3, 4, 6, 8,        10, 12, 14, 16, 18, 20, 21 or 22,    -   f) nucleic acid which is degenerated as a result of the genetic        code to a nucleotide sequence of a nucleic acid as given in any        of SEQ ID NOs 1, 2, 5, 7, 9, 11, 13, 15, 17 or 19 or as        defined (a) to (e),    -   g) nucleic acid which is diverging due to the differences in        codon usage between the organisms to a nucleotide sequence        encoding a polypeptide or protein as given in any of SEQ ID NOs        3, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21 or as defined in (a) to        (e),    -   h) nucleic acid which is diverging due to the differences in        alleles encoding a polypeptide or protein as given in any of SEQ        ID NOs, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21 or as defined        in (a) to (e),    -   i) nucleic acid encoding an immunologically active and/or        functional fragment of a polypeptide or protein encoded by a DNA        sequence as given in any of SEQ ID NOs 1, 2, 5, 7, 9, 11, 13,        15, 17 or 19 or as defined (a) to (e),    -   j) nucleic acid encoding a protein or polypeptide as defined in        SEQ ID Nos, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21 or as        defined in (a) to (i) characterized in that said sequence is        DNA, cDNA, genomic DNA or synthetic DNA.

It should be understood that the present invention also relates to anyof the nucleic acids defined in a) to j) for use in any of the methodsdescribed earlier.

The invention further relates to a nucleic acid molecule of at least 15nucleotides in length specifically hybridizing with, or specificallyamplifying one of the nucleic acids of the invention, preferably thosenucleic acids as defined in a) to j).

The invention also relates to a vector comprising a nucleic acid of theinvention, wherein said vector preferably is an expression vectorwherein the nucleic acid is operably linked to one or more controlsequences allowing the expression of said sequence in prokaryotic and/oreukaryotic host cells. As such the invention also relates to a host cellcontaining a nucleic acid or a vector of the invention. Said host cellcan be chosen from a bacterial, insect, fungal, yeast, plant or animalcell.

According to yet another embodiment, the invention relates to the use ofa natural or synthetic nucleic acid encoding a protein containing an “RSdomain” as defined earlier for use in any of the methods hereindescribed.

The invention also relates to the use of a natural or synthetic nucleicacid encoding a protein involved in the process of processing messengerRNA precursors in eukaryotic cells in any of the methods of theinvention.

The invention further relates to the use a nucleic acid with at least50% identity to at least one of the sequences herein described in amethod for enhancing stress tolerance comprising the manipulation of theprocess or pathway of processing mRNA precursors. Preferably saidnucleic acid originates from an eukaryotic cell or organism.

Also comprised within the invention are anti-sense moleculescorresponding to at least one of the nucleic acids of the invention andtheir use in any of the methods of the invention.

According to yet another embodiment, the present invention relates to apolypeptide encodable by at least one of the nucleic acids of theinvention, or a homologue thereof or a derivative thereof, or animmunologically active and/or functional fragment thereof, saidpolypeptide being natural, synthetic, enriched, isolated, cell-freeand/or recombinant. Each of these polypeptides can be used in any of themethods of the invention.

Preferred polypeptides are those comprising an amino acid sequence asgiven in any of SEQ ID NOs 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 21 or 22a homologue thereof or a derivative thereof, or an immunologicallyactive and/or functional fragment thereof.

The invention also relates to a method of producing a polypeptide of theinvention comprising culturing a host cell as described earlier underthe conditions allowing the expression of the polypeptide and recoveringthe produced polypeptide from the culture.

The invention also relates to a method for the production of transgenicplants, plant cells or plant tissues comprising the introduction of anucleic acid of the invention in an expressible format or a vector ofthe invention in said plant, plant cell or plant tissue.

The invention also relates to a method for the production of alteredplants, plant cells or plant tissues comprising the introduction of apolypeptide of the invention directly into a cell, a tissue or an organof said plant.

The invention further relates to a method for effecting the expressionof a polypeptide of the invention comprising the introduction of anucleic acid of the invention operably linked to one or more controlsequences or a vector of the invention stably into the genome of a plantcell.

The invention also relates to said methods for producing transgenicplants, further comprising regenerating a plant from said plant cell.

The invention further relates to a transgenic plant cell obtainable byone of the above-mentioned methods wherein said nucleic acid is stablyintegrated into the genome of said plant cell.

According to the invention, transgenic plants tolerant to salt stresscan be produced as a result of the expression of at least one of thenucleic acids of claim 13 or at least one of the polypeptides of claim25 or 26 or an anti-sense molecule of claim 23. Said transgenic plantsare also part of the invention, as well as transgenic plants which as aresult of the expression of at least one of the nucleic acids orantisense molecules of the invention or at least one of the polypeptidesof the invention show an alteration of their phenotype.

The invention relates to any harvestable part of a plant of theinvention which is preferably selected from the group consisting ofseeds, leaves, fruits, stem cultures, rhizomes, roots, tubers and bulbs.Also the progeny derived from any of the plants or plant parts of theinvention are part of the present invention.

According to another embodiment the invention relates to a method forenhancing stress tolerance in (a) plant(s) comprising expression of atleast one of the nucleic acids or at least one of the polypeptides ananti-sense molecule of the invention in cells, tissues or parts of saidplant(s).

According to another embodiment, the invention relates to a method foraltering stress tolerance in (a) plant(s) comprising expression of atleast one of the nucleic acids or at least one of the polypeptides or anant-sense molecule of the invention in cells, tissues or parts of saidplant(s).

It should be clear that the stress tolerance in the above methods canmean any stress caused by the environment such as, but not limited toosmotic stress, salt stress, drought stress, freezing stress or coldstress.

Furthermore, any of the methods, the nucleic acids, or antisensemolecules or the polypeptides of the invention can be used (in a method)for increasing yield, for stimulating growth which can be in any part ofthat plant, such as root, leave, seed.

The invention also relates to a plant obtainable by any of the abovedescribed methods for culturing on soil with high salt concentrations,preferably soils with a salt content of more than 1 mM salt ions.

Also forming part of the invention are new strains of yeast or otherunicellular eukaryotes more tolerant to salt stress as a result of theexpression of any of the nucleic acids and/or proteins of the invention.

The invention further relates to an in vitro cell culture systemcomprising animal, plant or host cells as defined earlier, tolerant tosalt, obtained as a result of the expression of at least one of thenucleic acids, vectors, polypeptides, or antisense molecule of claim 23.

The invention further relates to at least one therapeutic application inhumans derived from the methods described herein.

The invention also relates to an antibody specifically recognizing apolypeptide of the invention or a specific epitope thereof.

The invention further relates to a diagnostic composition comprising atleast a nucleic acid a vector, an antisense molecule, a polypeptide oran antibody of the invention.

DEFINITIONS AND ELABORATION TO THE EMBODIMENTS

Specific Definitions

“Manipulation of a process” herein means the interference with or themodulation of that process, preferably enhancing, catalysing, changingor altering that process. This interference can have an impact on everystep or every component or every product or every result of thatprocess. Also this interference can have an impact on the efficiency,the rate or the yield of that process.

“Genetic manipulation of a process” herein refers to the manipulation ofa process by any kind of interfering with the genetic sequences (e.g.nucleotide sequences, RNA, DNA) that are involved in that process. Nextto the natural genetic activity of the cell such as replication,transcription translation, and the processing of different nucleicacids, also molecular biology techniques and gentechnology techniquescomprised in the term “genetic manipulation of a process”. Theseartificial genetic techniques are know by the person skilled in the artand are for example cloning, transforming, recombining, expressing,overexpressing, silencing etc. As an example of “genetic manipulation ofa process” one can interfere with the genetic sequence encoding aprotein, which is involved in the process of processing messenger RNAprecursors. Alternatively, one can interfere with an RNA molecule (suchas a small nucleolar RNA), which is directly involved in the process ofprocessing messenger RNA precursors.

In the case that said genetic sequence encodes a protein and theexpression, the constitution, the structure or the location of thatcoding sequence is altered, the term “genetic manipulation of a protein”is used. More particularly one can alter the composition or theexpression of said coding sequences or one can introduce or delete saidcoding sequence in the host cell, which performs said process. Morespecifically said protein can be any component which is involved in theprocessing of pre-mRNA, such as but not limited to a component of theU1-snRNP or U2-snRNP complex, a transcription factor, an RNA bindingprotein or a nuclear movement protein.

“Biochemical manipulation of a process” herein refers to themanipulation of said process by using biochemical methods that interferewith said process. With biochemical methods is meant the use of anysubstance (chemicals, peptides, and molecules) which have an impact onbiological processes. For example one can introduce a peptide, or aprotein, or a biochemical or a chemical substance in the cell performingthat process, in order to interfere with that process. More particularlyone can introduce a protein or a peptide derived from the genes of thepresent invention directly into the cell, in order to enhance theprocess of processing messenger RNA precursors.

In the case that said biochemical method involves the use of a proteinor peptide or in case said biochemical method results in themodification of a protein, we use the term “biochemical manipulation ofa protein”.

“Cells” herein is to be taken in its broadest context and includes everyliving cell, such as prokaryotic and eukaryotic cells.

“Messenger RNA precursor” herein refers to any RNA molecule, which isnot yet operational as a mature messenger RNA, from which polypeptidescan be transcribed, because its composition, its structure or itslocation has to be changed. “Processing of messenger RNA precursors”herein refers to any process, which changes the composition, thestructure or the location of a messenger RNA precursor. Examples of suchprocesses in an eukaryotic cell are the synthesis of the messenger RNAprecursor during the transcription, the modification of the 5′ and/orthe 3′ ends of the precursor like polyadenylation, the splicing ofintrons from the precursor like the constitutive or the alternativesplicing, the metabolism of precursor or the translocation of theprecursor towards the nuclear envelop and the transport of the matureRNA to the cytoplams etc. Also the coupling of the different steps ofthis processing or metabolism of the precursor DNA are part of the term“processing of messenger RNA precursors” as used in this description.Post-transcriptional processing of precursor RNA comprises a complexpathway of endonucleolytic cleavages, exonucleolytic digestion andcovalent modifications. The general order of the various processingsteps is well conserved in eukaryotic cells, but the underlyingmechanisms are largely unknown. The pre-mRNA processing is an importantcellular activity and has been studied in the yeast cells Saccharomycescerevisiae by Venema and Tollervey (Yeast, (1995) 11(16): 1629-1650).The processing steps involve a variety of protein-protein interactionsas well as protein-RNA and RNA-RNA interactions. The precise role ofdifferent transcription factors, RNA binding proteins, and other nuclearproteins such as nuclear movement proteins and nuclear RNA's in theprocessing of pre-mRNA remains largely unknown. Therefor a proteininterfering with the process of processing messenger RNA precursors, canbe a protein of many different kinds. The nuclear transport of pre-mRNAinvolved several factors which can be used in the method of the presentinvention, since they are involved in the processing of pre-mRNA.Pre-mRNA is transcribed primarily from genes located at the interfacebetween chromatin domains and the interchromatin space. After partial orcomplete processing and complexing with nuclear proteins, thetranscripts leave their site of synthesis and travel through theinterchromatin space to the nuclear pores, where they are captured bythe export machinery for export to the cytoplasm. Transport-competentmRNA's are complexed with the correct complement of nuclear proteins(reviewed in Politz and Pederson, J. struct. Biol 2000, 129(2-3):252-257). The role of the U1-snRNP and the U2-snRNP is mainly situatedin the splicing of pre-mRNA. The product of the U1 small nuclearribonucleoprotein particle or complex U1-snRNP 70K (U1-70K) gene, a U1sn-RNP-specific protein, has been implicated in basic as well asalternative splicing or pre-mRNA in animals as well as in plants(Golovkin and Reddy, Plant Cell, 1996, (8): 1421-1435). In other reportsdifferent interacting proteins of the U1-70K protein are described, suchas for example SC35-like protein and serine/arginine-rich protein(Golovkin and Reddy, J. Biol Chem 1999, 245(51): 36428-36438). For theU2 small nuclear ribonucleoprotein auxiliary factors such as U2A havebeen described (Domon et al, J. Biol Chem 1998: 273(51): 34603-34610).Within the scope of the present invention are any proteins, which areinvolved in splicing of the messenger RNA-precursors. The role of smallnucleolar RNA's in the processing of pre-mRNA in plants has beendescribed (Brown and Shaw, the Plant Cell, 1998, 10: 649-657). Thereformanipulation of the process of processing pre-mRNA can also beestablished by interfering with RNA molecules. Jarrous et al (J. CellBiol. 1999, 146(3): 559-572) showed that Rpp29 and Rpp38 (proteinsubunits of human RNaseP) are found in the nucleolus and that theyreside in coiled bodies, organelles that are implicated in thebiogenesis of several other small nuclear ribonucleoproteins requiredfor the processing of precursor mRNA. These kinds of proteins which arepart of a Ribonucleoprotein Ribonuclease complex are involved in theprocessing of pre-mRNA can therefor also be used in the method of thepresent invention. The role of nuclear movement proteins is not wellestablished in the art. Still a lot of molecules involved in theprocessing of messenger RNA precursors remain to be elucidated.

“SR-like proteins” as used herein are described previously in inBlencowe et al. 1999, 77(4): 277-291: “The processing of messenger RNAprecursors (pre-mRNA) to mRNA requires a large number of proteins thatcontains domains rich in alternating arginine and serine residues (RSdomains). These include members of the SR family of splicing factors andproteins that are structurally and functionally distinct from the SRfamily collectively referred to below as SR-related proteins. Bothgroups of RS domain proteins function in constitutive and regulatedpre-mRNA splicing. Recently, several SR-related proteins have beenidentified that are associated with the transcriptional machinery. OtherSR-related proteins are associated with mRNA 3′ end formation and havebeen implicated in export”. The evidence that proteins containing RSdomains may play a fundamental role in the co-ordination of differentsteps in the synthesis and processing of pre-mRNA is further reviewed inBlencowe et al. 1999, 77(4): 277-291.

General Definitions

The terms “protein(s)”, “peptide(s)” or “oligopeptide(s)” orpolypeptide, when used herein refer to amino acids in a polymeric formof any length. Said terms also include known amino acid modificationssuch as disulphide bond formation, cysteinylation, oxidation,glutathionylation, methylation, acetylation, farnesylation,biotinylation, stearoylation, formylation, lipoic acid addition,phosphorylation, sulphation, ubiquitination, myristoylation,palmitoylation, geranylgeranylation, cyclization (e.g. pyroglutamic acidformation), oxidation, deamidation, dehydration, glycosylation (e.g.pentoses, hexosamines, N-acetylhexosamines, deoxyhexoses, hexoses,sialic acid etc.), acylation and radiolabels (e.g. ¹²⁵I, ¹³¹I, ³⁵S, ¹⁴C,³²P, ³³P, ³H) as well as non-naturally occurring amino acid residues,L-amino acid residues and D-amino acid residues.

“Homologues” or “Homologs ” of a protein of the invention are thosepeptides, oligopeptides, polypeptides, proteins and enzymes whichcontain amino acid substitutions, deletions andlor additions relative tothe said protein with respect to which they are a homologue withoutaltering one or more of its functional properties, in particular withoutreducing the activity of the resulting. For example, a homologue of saidprotein will consist of a bioactive amino acid sequence variant of saidprotein. To produce such homologues, amino acids present in the saidprotein can be replaced by other amino acids having similar properties,for example hydrophobicity, hydrophilicity, hydrophobic moment,antigenicity, propensity to form or break a-helical structures orβ-sheet structures, and so on.

Substitutional variants of a protein of the invention are those in whichat least one residue in said protein amino acid sequence has beenremoved and a different residue inserted in its place. Amino acidsubstitutions are typically of single residues, but may be clustereddepending upon functional constraints placed upon the polypeptide;insertions will usually be of the order of about 1-10 amino acidresidues and deletions will range from about 1-20 residues. Preferably,amino acid substitutions will comprise conservative amino acidsubstitutions, such as those described supra.

Insertional amino acid sequence variants of a protein of the inventionare those in which one or more amino acid residues are introduced into apredetermined site in said protein. Insertions can compriseamino-terminal and/or carboxy-terminal fusions as well as intra-sequenceinsertions of single or multiple amino acids. Generally, insertionswithin the amino acid sequence will be smaller than amino or carboxylterminal fusions, of the order of about 1 to 10 residues. Examples ofamino- or carboxy-terminal fusion proteins or peptides include thebinding domain or activation domain of a transcriptional activator asused in the yeast two-hybrid system, phage coat proteins,(histidine)₆-tag, glutathione S-transferase, protein A, maltose-bindingprotein, dihydrofolate reductase, Tag•100 epitope (EETARFQPGYRS), c-mycepitope (EQKLISEEDL), FLAG®-epitope (DYKDDDK), lacZ, CMP(calmodulin-binding peptide), HA epitope (YPYDVPDYA), protein C epitope(EDQVDPRLIDGK) and VSV epitope (YTDIEMNRLGK).

Deletional variants of a protein of the invention are characterised bythe removal of one or more amino acids from the amino acid sequence ofsaid protein.

Amino acid variants of a protein of the invention may readily be madeusing peptide synthetic techniques well known in the art, such as solidphase peptide synthesis and the like, or by recombinant DNAmanipulations. The manipulation of DNA sequences to produce variantproteins, which manifest as substitutional, insertional or deletionalvariants are well known in the art. For example, techniques for makingsubstitution mutations at predetermined sites in DNA having knownsequence are well known to those skilled in the art, such as by M13mutagenesis, T7-Gen in vitro mutagenesis kit (USB, Cleveland, Ohio),QuickChange Site Directed mutagenesis kit (Stratagene, San Diego,Calif.), PCR-mediated site-directed mutagenesis or other site-directedmutagenesis protocols. Another alternative to manipulate DNA sequencesto produce variant proteins, which manifest as substitutional,insertional or deletional variants comprises targeted in vivo genemodification which can be achieved by chimeric RNA/DNA oligonucleotidesas described by e.g. (Palmgren 1997;Yoon et al. 1996).

“Derivatives” of a protein of the invention are those peptides,oligopeptides, polypeptides, proteins and enzymes which comprise atleast about five contiguous amino acid residues of said polypeptide butwhich retain the biological activity of said protein. A “derivative” mayfurther comprise additional naturally-occurring, altered glycosylated,acylated or non-naturally occurring amino acid residues compared to theamino acid sequence of a naturally-occurring form of said polypeptide.Alternatively or in addition, a derivative may comprise one or morenon-amino acid substituents compared to the amino acid sequence of anaturally-occurring form of said polypeptide, for example a reportermolecule or other ligand, covalently or non-covalently bound to theamino acid sequence such as, for example, a reporter molecule which isbound thereto to facilitate its detection.

With “immunologically active” is meant that a molecule or specificfragments thereof such as epitopes or haptens are recognised by, i.e.bind to antibodies.

In the context of the current invention are also included homologous,derivatives and/or immunologically active fragments of any of theinventive polypeptides. “Antibodies” include monoclonal, polyclonal,synthetic or heavy chain camel antibodies as well as fragments ofantibodies such as Fab, Fv or scFv fragments. Monoclonal antibodies canbe prepared by the techniques as described previously e.g. (Liddle &Cryer 1991) which comprise the fusion of mouse myeloma cells to spleencells derived from immunised animals. The term “antibodies” furthermoreincludes derivatives thereof such as labelled antibodies. Antibodylabels include alkaline phosphatase, PKH2, PKH26, PKH67, fluorescein(FITC), Hoechst 33258, R-phycoerythrin (PE), rhodamine (TRITC), QuantumRed, Texas Red, Cy3, biotin, agarose, peroxidase, gold spheres andradiolabels (e.g. ¹²⁵I, ¹³¹I, ³⁵S, ¹⁴C, ³²P, ³³P, ³H). Tools inmolecular biology relying on antibodies against a protein includeprotein gel blot analysis, screening of expression libraries allowinggene identification, protein quantitative methods including ELISA andRIA, immunoaffinity purification of proteins, immunoprecipitation ofproteins e.g. (Magyar et al 1997) and immunolocalization. Other uses ofantibodies and especially of peptide antibodies include the study ofproteolytic processing (Loffler et al 1994; Woulfe et al. 1994),determination of protein active sites (Lerner 1982), the study ofprecursor and post-translational processing (Baron & Baltimore1982;Lerner et al 1981;Semler et al. 1982), identification of proteindomains involved in protein-protein interactions (Murakami et al. 1992)and the study of exon usage in gene expression (Tamura et al. 1991).

In the scope of the current invention are also antibodies recognisingthe proteins of the present invention or homologue, derivative orfragment thereof as defined supra.

The terms “gene(s)”, “polynucleotide(s)”, “nucleic acid, sequence(s)”,“nucleotide sequence(s)”, “DNA sequence(s)” or “nucleic acidmolecule(s)”, when used herein refer to nucleotides, eitherribonucleotides or deoxytibonucleobdes or a combination of both, in apolymeric form of any length. Said terms furthermore includedouble-stranded and single-stranded DNA and RNA. Said terms also includeknown nucleotide modifications such as methylation, cyclization and‘caps’ and substitution of one or more of the naturally occurringnucleotides with an analogue such as inosine. Said terms also encompasspeptide nucleic acids (PNAs), a DNA analogue in which the backbone is apseudopeptid e consisting of N-(2-aminoethyl)-glycine units rather thana sugar. PNAs mimic the behaviour of DNA and bind complementary nucleicacid strands. The neutral backbone of PNA results in stronger bindingand greater specificity than normally achieved. In addition, the uniquechemical, physical and biological properties of PNA have been exploitedto produce powerful biomolecular tools, anti-sense and anti-gene agents,molecular probes and biosensors.

With “recombinant DNA molecule” or “chimeric gene” is meant a hybrid DNAproduced by joining pieces of DNA from different sources. With“heterologous nucleotide sequence” is intended a sequence that is notnaturally occurring with the promoter sequence. While this nucleotidesequence is heterologous to the promoter sequence, it may be homologous,or native, or heterologous, or foreign, to the plant host. “Sensestrand” refers to the strand of a double-stranded DNA molecule that ishomologous to a mRNA transcript thereof. The “anti-sense strand”contains an inverted sequence, which is complementary to that of the“sense strand”.

A “coding sequence” or “open reading frame” or “ORF” is defined as anucleotide sequence that can be transcribed into mRNA and/or translatedinto a polypeptide when placed under the control of appropriateregulatory sequences, i.e. when said coding sequence or ORF is presentin an expressible format. Said coding sequence of ORF is bounded by a 5′translation start codon and a 3′ translation stop codon. A codingsequence or ORF can include, but is not limited to RNA, mRNA, cDNA,recombinant nucleotide sequences, synthetically manufactured nucleotidesequences or genomic DNA. Said coding sequence or ORF can be interruptedby intervening nucleic acid sequences. Genes and coding sequencesessentially encoding the same protein but isolated from differentsources can consist of substantially divergent nucleic acid sequences.Reciprocally, substantially divergent nucleic acid sequences can bedesigned to effect expression of essentially the same protein. Saidnucleic acid sequences are the result of e.g. the existence of differentalleles of a given gene, or of the degeneracy of the genetic code or ofdifferences in codon usage. Thus amino acids such as methionine andtryptophan are encoded by a single codon whereas other amino acids suchas arginine, leucine and serine can each be translated from up to sixdifferent codons. Differences in preferred codon usage are illustratedbelow for Agrobacterium tumefaciens (a bacterium), A. thaliana, M.sativa (two dicotyledonous plants) and Oryza sativa (a monocotyledonousplant). These examples were extracted from (www.kazusa.orjp/codon). Togive one example, the codon GGC (for glycine) is the most frequentlyused codon in A. tumefaciens (36.2%), is the second most frequently usedcodon in O. sativa but is used at much lower frequencies in A. thalianaand M. sativa (9% and 8.4%, respectively). Of the four possible codonsencoding glycine (see Table 2), said GGC codon is most preferably usedin A. tumefaciens and O. sativa. However, in A. thaliana this is the GGA(and GGU) codon whereas in M. sativa this is the GGU (and GGA) codon.Allelic variants are further defined as to comprise single nucleotidepolymorphisms (SNPs) as well as small insertionideletion polymorphisms(INDELs; the size of INDELs is usually less than 100. bp). SNPs andINDELs form the largest set of sequence variants in naturally occurringpolymorphic strains of most organisms. They are helpful in mapping genesand discovery of genes and gene functions. They are furthermore helpfulin identification of genetic loci, e.g. plant genes, involved indetermining processes such as growth rate, plant size and plant yield,plant vigor, disease resistance, stress tolerance etc. Many techniquesare nowadays available to identify SNPs and/or INDELs including (i) PCRfollowed by denaturing high performance liquid chromatography (DHPLC;e.g. . (Cho et al. 1999)); (ii) constant denaturant capillaryelectrophoresis (CDCE) combined with high-fidelity PCR (e.g.(U-Sucholeiki et al. 1999)); (iii) denaturing gradient gelelectrophoresis (e.g. Fischer and Lerman 1983); (iv) matrix-assistedlaser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS; e.g. (Ross et al. 2000)); (v) real-time fluorescence monitoring PCRassays (e.g. Tapp et al. 2000); (vi) Acrydite.TM. gel technology (e.g.Kenney et al. 1998); (vii) cycle dideoxy fingerprinting (CddF; e.g.(Langemeier et al. 1994); (viii) single-strand conformation polymorphism(SSCP) analysis (e.g. (Vidal-Puig & Moller 1994)) and (ix)mini-sequencing primer extension reaction (e.g. Syvanen 1999). Thetechnique of ‘Targeting Induced Local Lesions in Genomes’ (TILLING:(McCallum et al. 2000a;McCallum et al. 2000b)), which Is a variant of(i)supra, can also be applied to rapidly identify an altered gene in e.g.chemically mutagenized plant individuals showing interesting phenotypes.

“Hybridisation” is the process wherein substantially homologouscomplementary nucleotide sequences anneal to each other. Thehybridisation process can occur entirely in solution, i.e. bothcomplementary nucleic acids are in solution. Tools in molecular biologyrelying on such a process include the polymerase chain reaction (PCR;and all methods based thereon), subtractive hybridisation, random primerextension, nuclease S1 mapping, primer extension, reverse transcription,cDNA synthesis, differential display of RNAs, and DNA sequencedetermination. The hybridisation process can also occur with one of thecomplementary nucleic acids immobilised to a matrix such as magneticbeads, Sepharose beads or any other resin. Tools in molecular biologyrelying on such a process include the isolation of poly (A+) mRNA. Thehybridisation process can furthermore occur with one of thecomplementary nucleic acids immobilised to a solid support such as anitro-cellulose or nylon membrane or immobilised by e.g.photolithography to e.g. a siliceous glass support (the latter known asnucleic acid arrays or microarrays or as nucleic acid chips). Tools inmolecular biology relying on such a process include RNA and DNA gel blotanalysis, colony hybridisation, plaque hybridisation, in situhybridisation and microarray hybridisation. In order to allowhybridisation to occur, the nucleic acid molecules are generallythermally or chemically denatured to melt a double strand into twosingle strands and/or to remove hairpins or other secondary structuresfrom single stranded nucleic acids. The stringency of hybridisation isinfluenced by conditions such as temperature, salt concentration andhybridisation buffer composition. High stringency conditions forhybridisation include high temperature and/or low salt concentration(salts include NaCl and Na₃-citrate) and/or the inclusion of formamidein the hybridisation buffer and/or lowering the concentration ofcompounds such as SDS (detergent) in the hybridisation buffer and/orexclusion of compounds such as dextran sulphate or polyethylene glycol(promoting molecular crowding) from the hybridisation buffer.Conventional hybridisation conditions are described e.g. (Sambrook etal. 1989) but the skilled craftsman will appreciate that numerousdifferent hybridisation conditions can be designed in function of theknown or the expected homology and/or length of the nucleic acidsequence. With specifically hybridising is meant hybridising understringent conditions. Sufficiently low stringency hybridisationconditions are particularly preferred to isolate nucleic acidsheterologous to the DNA sequences of the invention defined supra.Elements contributing to said heterology include allelism, degenerationof the genetic code and differences in preferred codon usage asdiscussed supra.

Accordingly, the scope of the current invention is also related to theuse of the inventive DNA sequences encoding the polypeptides of thepresent invention, homologue, derivative and/or immunologically fragmentthereof as defined higher in any method of hybridisation. The currentinvention furthermore also relates to DNA sequences hybridising to saidinventive DNA sequences.

“Specifically amplifying” herein using an amplification method which isselective and only amplifies a nucleic acid sequence with a specificbase-pair composition (e.g. polymerase chain reaction).

DNA sequences as defined in the current invention can be interrupted byintervening sequences. With “intervening sequences” is meant any nucleicacid sequence which disrupts a coding sequence comprising said inventiveDNA sequence or which disrupts the expressible format of a DNA sequencecomprising said inventive DNA sequence. Removal of the interveningsequence restores said coding sequence or said expressible format.Examples of intervening sequences include introns, mobilizable DNAsequences such as transposons and DNA tags such as e.g. a T-DNA. With“mobilizable DNA sequence” is meant any DNA sequence that can bemobilised as the result of a recombination event.

To effect expression of a protein in a cell, tissue or organ, preferablyof plant origin, either the protein may be introduced directly to saidcell, such as by microinjection or ballistic means or alternatively, anisolated nucleic acid molecule encoding said protein may be introducedinto said cell, tissue or organ in an expressible format.

Preferably, the DNA sequence of the invention comprises a codingsequence or open reading frame (ORF) encoding a protein of the presentinvention or a homologue or derivative thereof or an immunologicallyactive thereof as defined supra. The preferred proteins of the inventioncomprises the amino acid sequence as presented in SEQ ID NO. 3, 4, 6, 8,10, 12, 14, 16, 18, 20 and 21.

With “vector” or “vector sequence” is meant a DNA sequence, which can beintroduced in an organism by transformation and can be stably maintainedin said organism. Vector maintenance is possible in e.g. cultures ofEscherichia coli, A. tumefaciens, Saccharomyces cerevisiae orSchizosaccharomyces pombe. Other vectors such as phagemids and cosmidvectors can be maintained and multiplied in bacteria and/or viruses.Vector sequences generally comprise a set of unique sites recognised byrestriction enzymes, the multiple cloning site (MCS), wherein one ormore non-vector sequence(s) can be inserted.

With “non-vector sequence” is accordingly meant a DNA sequence which isintegrated in one or more of the sites of the MCS comprised within avector.

“Expression vectors” form a subset of vectors which, by virtue ofcomprising the appropriate regulatory sequences enabling the creation ofan expressible format for the inserted non-vector sequence(s), thusallowing expression of the protein encoded by said non-vectorsequence(s). Expression vectors are known in the art enabling proteinexpression in organisms including bacteria (e.g. E. coli), fungi (e.g.S. cerevisiae, S. pombe, Pichia pastoris), insect cells (e.g.baculoviral expression vectors), animal cells (e.g. COS or CHO cells)and plant cells (e.g. potato virus X-based expression vectors, see e.g.Vance et al. 1998—WO9844097). See also further in this specification fortypical plant expression vectors.

The current invention clearly includes any vector or expression vectorcomprising a non-vector DNA sequence comprising the nucleotide sequencesaccording to the present invention or a non-vector sequence encoding theproteins of the present invention, or the homologue, derivative and/orimmunologically active fragment thereof as defined supra.

As an alternative to expression vector-mediated protein production inbiological systems, chemical protein synthesis can be applied. Syntheticpeptides can be manufactured in solution phase or in solid phase. Solidphase peptide synthesis (Merrifield 1963) is, however, the most commonway and involves the sequential addition of amino acids to create alinear peptide chain.

By “expressible format” or “under the control of expression controlsequences” is meant that the isolated nucleic acid molecule is in a formsuitable for being transcribed into mRNA and/or translated to produce aprotein, either constitutively or following induction by anintracellular or extracellular signal, such as an environmental stimulusor stress (mitogens, anoxia, hypoxia, temperature, salt, light,dehydration, etc) or a chemical compound such as IPTG(isopropyl-β-D-thiogalactopyranoside) or such as an antibiotic(tetracycline, ampicillin, rifampicin, kanamycin), hormone (e.g.gibberellin, auxin, cytokinin, glucocorticoid, brassinosteroid,ethylene, abscisic acid etc), hormone analogue (iodoacetic acid (IAA),2,4-D, etc), metal (zinc, copper, iron, etc), or dexamethasone, amongstothers. As will be known to those skilled in the art, expression of afunctional protein may also require one or more post-translationalmodifications, such as glycosylation, phosphorylation,dephosphorylation, or one or more protein-protein interactions, amongstothers. All such processes are included within the scope of the term“expressible format”.

Preferably, expression of a protein in a specific cell, tissue, ororgan, preferably of plant origin, is effected by introducing andexpressing an isolated nucleic acid molecule encoding said protein, suchas a cDNA molecule, genomic gene, synthetic oligonucleotide molecule,mRNA molecule or open reading frame, to said cell, tissue or organ,wherein said nucleic acid molecule is placed operably in connection withsuitable regulatory sequences including a promoter, preferably aplant-expressible promoter, and a terminator sequence.

“Regulatory sequence” refers to control DNA sequences, which arenecessary to affect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism. In prokaryotes, control sequences generally includepromoters, ribosomal binding sites, and terminators. In eukaryotesgenerally control sequences include promoters, terminators and enhancersor silencers.

Within the scope of the invention are also the nucleotide sequences asdefined in the present invention fused to any regulatory sequence. Theterm “control sequences” is intended to include, at a minimum, allcomponents the presence of which are necessary for expression, and mayalso include additional advantageous components and which determineswhen, how much and where a specific gene is expressed.

Reference herein to a “promoter” is to be taken in its broadest contextand includes the transcriptional regulatory sequences derived from aclassical eukaryotic genomic gene, including the TATA box which isrequired for accurate transcription initiation, with or without a CCMTbox sequence and additional regulatory elements (i.e. upstreamactivating sequences, enhancers and silencers) which alter geneexpression in response to developmental and/or external stimuli, or in atissue-specific manner.

The term “promoter” also includes the transcriptional regulatorysequences of a classical prokaryotic gene, in which case it may includea −35 box sequence and/or a −10 box transcriptional regulatorysequences.

The term “promoter” is also used to describe a synthetic or fusionmolecule or derivative, which confers, activates or enhances expressionof a nucleic acid molecule in a cell, tissue or organ.

Promoters may contain additional copies of one or more specificregulatory elements, to further enhance expression and/or to alter thespatial expression and/or temporal expression of a nucleic acid moleculeto which it is operably connected. Such regulatory elements may beplaced adjacent to a heterologous promoter sequence to drive expressionof a nucleic acid molecule in response to e.g. copper, glucocorticoids,dexamethasone, tetracycline, gibberellin, cAMP, abscisic acid, auxin,wounding, ethylene, jasmonate or salicylic acid or to confer expressionof a nucleic acid molecule to specific cells, tissues or organs such asmeristems, leaves, roots, embryo, flowers, seeds or fruits. In thecontext of the present invention, the promoter preferably is aplant-expressible promoter sequence. Promoters, however, that alsofunction or solely function in non-plant cells such as bacteria, yeastcells, insect cells and animal cells are not excluded from theinvention. By “plant-expressible” is meant that the promoter sequence,including any additional regulatory elements added thereto or containedtherein, is at least capable of inducing, conferring, activating orenhancing expression in a plant cell, tissue or organ, preferably amonocotyledonous or dicotyledonous plant cell, tissue, or organ.

The terms “plant-operable” and “operable in a plant” when used herein,in respect of a promoter sequence, shall be taken to be equivalent to aplant-expressible promoter sequence.

In the present context, a “regulated promoter” or “regulatable promotersequence” is a promoter that is capable of conferring expression on astructural gene in a particular cell, tissue, or organ or group ofcells, tissues or organs of a plant, optionally under specificconditions, however does generally not confer expression throughout theplant under all conditions. Accordingly, a regulatable promoter sequencemay be a promoter sequence that confers expression on a gene to which itis operably connected in a particular location within the plant oralternatively, throughout the plant under a specific set of conditions,such as following induction of gene expression by a chemical compound orother elicitor. Preferably, the regulatable promoter used in theperformance of the present invention confers expression in a specificlocation within the plant, either constitutively or following induction,however not in the whole plant under any circumstances. Included withinthe scope of such promoters are cell-specific promoter sequences,tissue-specific promoter sequences, organ-specific promoter sequences,cell cycle specific gene promoter sequences, inducible promotersequences and constitutive promoter sequences that have been modified toconfer expression in a particular part of the plant at any one time,such as by integration of said constitutive promoter within atransposable genetic element (Ac, Ds, Spm, En, or other transposon).Those skilled in the art will be aware that an “inducible promoter” is apromoter the transcriptional activity of which is increased or inducedin response to a developmental, chemical, environmental, or physicalstimulus. Within the scope of the present invention are the nucleotidesequences as defined in the claims, fused to a stress induciblepromoter. Similarly, the skilled craftsman will understand that a“constitutive promoter” is a promoter that is transcriptionally activethroughout most, but not necessarily all parts of an organism,preferably a plant, during most, but not necessarily all phases of itsgrowth and development. Contrarily the term “ubiquitous promoter” istaken to indicate a promoter that is transcriptionally active throughoutmost, but not necessarily all parts of an organism, preferably a plant.

Those skilled in the art will readily be capable of selectingappropriate promoter sequences for use in regulating appropriateexpression of the proteins of the present inventio as described suprafrom publicly-available or readily-available sources, without undueexperimentation.

Placing a nucleic acid molecule under the regulatory control of apromoter sequence, or in operable connection with a promoter sequencemeans positioning said nucleic acid molecule such that expression iscontrolled by the promoter sequence. A promoter is usually, but notnecessarily, positioned upstream, or at the 5′-end, and within 2 kb ofthe start site of transcription, of the nucleic acid molecule which itregulates. In the construction of heterologous promoter/structural genecombinations it is generally preferred to position the promoter at adistance from the gene transcription start site that is approximatelythe same as the distance between that promoter and the gene it controlsin its natural setting (i.e., the gene from which the promoter isderived).

“Expression” means the production of a protein or nucleotide sequence inthe cell itself or in a cell-free system. It includes transcription intoan RNA product, post-transcriptional modification and/or translation toa protein product or polypeptide from a DNA encoding that product, aswell as possible post-translational modifications.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A control sequence “operably linked” to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the control sequences. Incase the control sequence is a promoter, it is obvious for a skilledperson that double-stranded nucleic acid is preferably used.

The term “terminator” refers to a DNA sequence at the end of atranscriptional unit which signal termination of transcription.Terminators are 3′-non-translated DNA sequences containing apolyadenylation signal, which facilitates the addition of polyadenylatesequences to the 3′-end of a primary transcript. Terminators active incells derived from viruses, yeasts, moulds, bacteria, insects, birds,mammals and plants are known and described in the literature. They maybe isolated from bacteria, fungi, viruses, animals and/or plants.

Examples of terminators particularly suitable for use in the geneconstructs of the present invention include the Agrobacteriumtumefaciens nopaline synthase (NOS) gene terminator, the Agrobacteriumtumefaciens octopine synthase (OCS) gene terminator sequence, theCauliflower mosaic virus (CaMV) 35S gene terminator sequence, the Oryzasativa ADP-glucose pyrophosphorylase terminator sequence (t3′Bt2), theZea mays zein gene terminator sequence, the rbcs-1A gene terminator, andthe rbcs-3A gene terminator sequences, amongst others.

Those skilled in the art will be aware of suitable promoter sequencesand terminator sequences which may be suitable for use in performing theinvention. Such sequences may readily be used without any undueexperimentation.

Altering the expression of e gene can be the downregulation ofexpression. In the context of the current invention is envisaged thedownregulation of the expression of some proteins which are involved inthe process of processing of messenger RNA precursors.

This can for example result in a beneficial effect on messenger RNAprocessing and stress tolerance, when said protein was a negativeregulator of the process. “Downregulation of expression” as used hereinmeans lowering levels of gene expression and/or levels of active geneproduct and/or levels of gene product activity. Decreases in expressionmay be accomplished by e.g. the addition of coding sequences or partsthereof in a sense orientation (if resulting in co-suppression) or in anantisense orientation relative to a promoter sequence and furthermore bye.g. insertion mutagenesis (e.g. T-DNA insertion or transposoninsertion) or by gene silencing strategies as described by e.g. Angelland Baulcombe 1998 (WO9836083), Lowe et al. 1989 (WO9853083), Lederer etal. 1999 (WO9915682) or Wang et al. 1999 (WO9953050). Genetic constructsaimed at silencing gene expression may have the nucleotide sequence ofsaid gene (or one or more parts thereof) contained therein in a senseand/or antisense orientation relative to the promoter sequence. Anothermethod to downregulate gene expression comprises the use of ribozymes,e.g. as described in Atkins et al. 1994 (WO9400012), Lenee et al. 1995(WO9503404), Lutziger et al. 2000 (WO00009619), Prinsen et al. 1997(WO9713865) and Scott et al. 1997 (WO9738116).

Modulating, including lowering, the level of active gene products or ofgene product activity can be achieved by administering or exposingcells, tissues, organs or organisms to said gene product, a homologue,analogue, derivative and/or immunologically active fragment thereof.Immunomodulation is another example of a technique capable ofdownregulation levels of active gene product and/or of gene productactivity and comprises administration of or exposing to or expressingantibodies to said gene product to or in cells, tissues, organs ororganisms wherein levels of said gene product and/or gene productactivity are to be modulated. Such antibodies comprise “plantibodies”,single chain antibodies, IgG antibodies and heavy chain camel antibodiesas well as fragments thereof. Within the scope of the present inventionare antibodies, recognizing the proteins of the present invention andthat can be used for said immunomodulation.

Modulating, including lowering, the level of active gene products or ofgene product activity can furthermore be achieved by administering orexposing cells, tissues, organs or organisms to an inhibitor oractivator of said gene product or the activity thereof. Such inhibitorsor activators include proteins (comprising e.g. proteinases and kinases)and chemical compounds identified according to the current

By “cell fate and/or plant development and/or plant morphology and/orbiochemistry and/or physiology” is meant that one or more developmentaland/or morphological and/or biochemical and/or physiologicalcharacteristics of a plant is altered by the performance of one or moresteps pertaining to the invention described herein.

“Cell fate” refers to the cell-type or cellular characteristics of aparticular cell that are produced during plant development or a cellularprocess therefor, in particular during the cell cycle or as aconsequence of a cell cycle process.

“Plant development” or the term “plant developmental characteristic” orsimilar term shall, when used herein, be taken to mean any cellularprocess of a plant that is involved in determining the developmentalfate of a plant cell, in particular the specific tissue or organ typeinto which a progenitor cell will develop. Cellular processes relevantto plant development will be known to those skilled in the art. Suchprocesses include, for example, morphogenesis, photomorphogenesis, shootdevelopment, root development, vegetative development, reproductivedevelopment, stem elongation, flowering, and regulatory mechanismsinvolved in determining cell fate, in particular a process or regulatoryprocess involving the cell cycle.

“Plant morphology” or the term “plant morphological characteristic” orsimilar term will, when used herein, be understood by those skilled inthe art to refer to the external appearance of a plant, including anyone or more structural features or combination of structural featuresthereof. Such structural features include the shape, size, number,position, colour, texture, arrangement, and patternation of any cell,tissue or organ or groups of cells, tissues or organs of a plant,including the root, stem, leaf, shoot, petiole, trichome, flower, petal,stigma, style, stamen, pollen, ovule, seed, embryo, endosperm, seedcoat, aleurone, fibre, fruit, cambium, wood, heartwood, parenchyma,aerenchyma, sieve element, phloem or vascular tissue, amongst others.

“Plant biochemistry” or the term “plant biochemical characteristic” orsimilar term will, when used herein, be understood by those skilled inthe art to refer to the metabolic and catalytic processes of a plant,including primary and secondary metabolism and the products thereof,including any small molecules, macromolecules or chemical compounds,such as but not limited to starches, sugars, proteins, peptides,enzymes, hormones, growth factors, nucleic acid molecules, celluloses,hemicelluloses, calloses, lectins, fibres, pigments such asanthocyanins, vitamins, minerals, micronutrients, or macronutrients,that are produced by plants.

“Plant physiology” or the term “plant physiological characteristic” orsimilar term will, when used herein, be understood to refer to thefunctional processes of a plant, including developmental processes suchas growth, expansion and differentiation, sexual development, sexualreproduction, seed set, seed development, grain filling, asexualreproduction, cell division, dormancy, germination, light adaptation,photosynthesis, leaf expansion, fiber production, secondary growth orwood production, amongst others; responses of a plant toexternally-applied factors such as metals, chemicals, hormones, growthfactors, environment and environmental stress factors (eg. anoxia,hypoxia, high temperature, low temperature, dehydration, light, daylength, flooding, salt, heavy metals, amongst others), includingadaptive responses of plants to said externally-applied factors.

The term “environmental stress” has been defined in different ways inthe prior art and largely overlaps with the term “osmotic stress”.(Holmberg & Bülow, 1998, Trends plant sci. 3, 61-66) for instance definedifferent environmental stress factors which result in abiotic stress.The term osmotic stress as used herein is meant as a stress situationinduces by conditions as salinity, drought, heat, chilling (or cold) andfreezing. With The term “environmental stress” as used in the presentinvention refers to any adverse effect on metabolism, growth orviability of the cell, tissue, seed, organ or whole plant which isproduced by an non-living or non-biological environmental stress. Moreparticularly, it also encompasses environmental factors such as waterstress (flooding, water logging, drought, dehydration), anaerobic (lowlevel of oxygen, CO₂ etc.), aerobic stress, osmotic stress, salt stress,temperature stress (hot/heat, cold, freezing, frost) or nutrientsdeprivation, pollutants stress (heavy metals, toxic chemicals), ozone,high light, pathogen (including viruses, bacteria, fungi, insects andnematodes) and combinations of these.

The term “anaerobic stress” means any reduction in oxygen levelssufficient to produce a stress as herein before defined, includinghypoxia and anoxia.

The term “flooding stress” refers to any stress which is associated withor induced by prolonged or transient immersion of a plant, plant part,tissue or isolated cell in a liquid medium such as occurs duringmonsoon, wet season, flash flooding or excessive irrigation of plants,etc.

“Cold stress” or “chilling stress” and “heat stress” are stressesinduced by temperatures that are respectively, below or above, theoptimum range of growth temperatures for a particular plant species.Such optimum growth temperature ranges are readily determined or knownto those skilled in the art.

“Dehydration stress” is any stress which is associated with or inducedby the loss of water, reduced turgor or reduced water content of a cell,tissue, organ or whole plant.

“Drought stress” refers to any stress, which is induced by or associatedwith the deprivation of water or reduced supply of water to a cell,tissue, organ or organism.

“Oxidative stress” refers to any stress, which increases theintracellular level of reactive oxygen species.

The terms “salinity-induced stress”, “salt stress” or “salt ionictoxicity” or mineral salt toxicity or similar terms refer to any stresswhich is associated with or induced by elevated concentrations of saltand which result in a perturbation in the osmotic potential of theintracellular or extracellular environment of a cell. The best knownexamples of mineral salts that induce said salt stress are Na+ and Li+.

The transgenic plants obtained in accordance with the method of thepresent invention, upon the presence of the polynucleic acid and/orregulatory sequence introduced into said plant, attain resistance,tolerance or improved tolerance or resistance against environmentalstress which the corresponding wild-type plant was susceptible to.

The terms “tolerance” and “resistance” cover the range of protectionfrom a delay to complete inhibition of alteration in cellularmetabolism, reduced cell growth and/or cell death caused by theenvironmental stress conditions defined herein before. Preferably, thetransgenic plant obtained in accordance with the method of the presentinvention is tolerant or resistant to environmental stress conditions inthe sense that said plant is capable of growing substantially normalunder environmental conditions where the corresponding wild-type plantshows reduced growth, metabolism, viability, productivity and/or male orfemale sterility. As used herein, “stress tolerance” refers to thecapacity to grow and produce biomass during stress, the capacity toreinitiate growth and biomass production after stress, and the capacityto survive stress. The term “stress tolerance” also covers the capacityof the plant to undergo its developmental program during stresssimilarly to under non-stressed conditions, e.g. to switch from dormancyto germination and from vegetative to reproductive phase under stressedconditions similarly as under non-stressed conditions. Methodologies todetermine plant growth or response to stress include, but are notlimited to height measurements, leaf area, plant water relations,ability to flower, ability to generate progeny and yield or any othermethodology known to those skilled in the art.

“Growth” refers to the capacity of the plant or of plant parts to growand increase in biomass while “yield” refers to the harvestable biomassof plants or plant parts, particularly those parts of commercial value.“Growth and/or yield under stressed and non-stressed conditions” refersto the fact that field-grown plants almost always will experience someform of stress, albeit mild. It is therefore preferred not todistinguish non-stressed from mild-stressed conditions. As certainbeneficial effects of the invention on growth and yield are expected tooccur under both severe and mild stress conditions, they are thusdescribed as increasing growth and/or yield under stressed andnon-stressed conditions. Means for introducing recombinant DNA intoplant tissue or cells include, but are not limited to, transformationusing CaCl₂ and variations thereof, in particular the method describedpreviously (Hanahan 1983), direct DNA uptake into protoplasts (Krens etal. 1982; Paszkowski et al. 1984), PEG-mediated uptake to protoplasts(Armstrong et al. 1990) microparticle bombardment, electroporation(Fromm et al. 1985), microinjection of DNA (Crossway et al. 1986; Frommet al. 1985), microparticle bombardment of tissue explants or cells(Christou et al. 1988), vacuum-infiltration of tissue with nucleic acid,or in the case of plants, T-DNA-mediated transfer from Agrobacterum tothe plant tissue as described essentially (An et al 1985;Dodds1985;Herrera-Estrella et al. 1983a;Herrera-Estrella et al. 1983b).Methods for transformation of monocotyledonous plants are well known inthe art and include Agrobacterium-mediated transformation (Cheng et al.1997—WO9748814; Hansen 1998—WO9854961, Hiei et al. 1994—WO9400977;Hieiet al. 1998—WO9817813;Rikiishi et al. 1999—WO9904618; Saito et al.1995—WO9506722), microprojectile bombardment (Adams et al. 1999—U.S.Pat. No. 5,969,213; Bowen et al. 1998 —U.S. Pat. No. 5,736,369; Chang etal. 1994—WO9413822; Lundquist et al. 1999—U.S. Pat. Nos.5,874,265/5,990,390; Vasil and Vasil 1995—U.S. Pat. No. 5,405,765;Walker et al. 1999—U.S. Pat. No. 5,955,362), DNA uptake (Eyal et al.1993—WO9318168), microinjection of Agrobacterium cells (von Holt1994—DE4309203) and sonication (Finer et al. 1997—U.S. Pat. No.5693512).

A whole plant may be regenerated from the transformed or transfectedcell, in accordance with procedures well known in the art. Plant tissuecapable of subsequent clonal propagation, whether by organogenesis orembryogenesis, may be transformed with a gene construct of the presentinvention and a whole plant regenerated therefrom. The particular tissuechosen will vary depending on the clonal propagation systems availablefor, and best suited to, the particular species being transformed.Exemplary tissue targets include leaf disks, pollen, embryos,cotyledons, hypocotyls, megagametophytes, callus tissue, existingmeristematic tissue (e.g., apical meristem, axillary buds, and rootmeristems), and induced meristem tissue (e.g., cotyledon meristem andhypocotyl meristem).

Preferably, the plant is produced according to the inventive method istransfected or transformed with a genetic sequence, or amenable to theintroduction of a protein, by any art-recognized means, such asmicroprojectile bombardment, microinjection, Agrobacterium-mediatedtransformation (including the ‘flower dip’ transformation method;(Bechtold & Pelletier 1998;Trieu et al. 2000)), protoplast fusion, orelectroporation, amongst others. Most preferably said plant is producedby Agrobacteriuim-mediated transformation.

With “binary transformation vector” is meant a T-DNA transformationvector comprising: a T-DNA region comprising at least one gene ofinterest and/or at least one selectable marker active in the eukaryoticcell to be transformed; and a vector backbone region comprising at leastorigins of replication active in E. coli and Agrobacterium and markersfor selection in E. coli and Agrobacterium. Alternatively, replicationof the binary transformation vector in Agrobacterium is dependent on thepresence of a separate helper plasmid. The binary vector pGreen and thehelper plasmid pSoup form an example of such a system as described ine.g. (Hellens et al. 2000) or as available on the internet sitewww.pgreen.ac.uk.

The T-DNA borders of a binary transformation vector can be derived fromoctopine-type or nopaline-type Ti plasmids or from both. The T-DNA of abinary vector is only transferred to a eukaryotic cell in conjunctionwith a helper plasmid. Also known in the art are multiple binary vectorAgrobacterium strains for efficient co-transformation of plants (Bidneyand Scelonge 2000—WO001 8939).

“Host” or “host cell” or host organism” herein is any prokaryotic oreukaryotic cell or organism that can be a recipient of the sequences ofthe present invention. A “host,” as the term is used herein, includesprokaryotic or eukaryotic organisms that can be genetically engineered.For examples of such hosts, see Maniatis et al., Molecular Cloning. ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1982). “plant cell” comprises any cell derived from any plant andexisting in culture as a single cell, a group of cells or a callus. Aplant cell may also be any cell in a developing or mature plant inculture or growing in nature.

“Plant” or “Plants” comprise all plant species which belong to thesuperfamily Viridiplantae. The present invention is applicable to anyplant, in particular a monocotyledonous plants and dicotyledonous plantsincluding a fodder or forage legume, ornamental plant, food crop, tree,or shrub selected from the list comprising Acacia spp., Acer spp.,Actinidia spp., Aesculus spp., Agathis australis, Albizia amara,Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Asteliafragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassicaspp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadabafarinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicumspp., Cassia spp., Centroema pubescens, Chaenomeles spp., Cinnamomumcassia, Coffea arabica, Colophospermum mopane, Coronillia varia,Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp.,Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogonspp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davalliadivaricata, Desmodium spp., Dicksonia squarosa, Diheteropogonamplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloapyramidalis, Ehrartia spp., Eleusine coracana, Eragrestis spp.,Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia villosa,Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flemingia spp,Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtiacoleosperma, Hedysarum spp., Hemarthia altissima, Heteropogon contortus,Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypertheliadissoluta, Indigo incarnata, Iris spp., Leptarrhena pyrolifolia,Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex,Lotonus bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihotesculenta, Medicago sativa, Metasequoia glyptostroboides, Musasapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryzaspp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petuniaspp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photiniaspp., Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara,Pogonarthria fleckii, Pogonarthria squarrosa, Populus spp., Prosopiscineraria, Pseudotsuga menziesii, Pterolbium stellatum, Pyrus communis,Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhusnatalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosaspp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitysverticillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghumbicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides,Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themedatriandra, Trifollum spp., Triticum spp., Tsuga heterophylla, Vacciniumspp., Vicia spp. Vitis vinifera, Watsonia pyramidata, Zantedeschiaaethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, brusselsprout, cabbage, canola, carrot, cauliflower, celery, collard greens,flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean,straw, sugarbeet, sugar cane, sunflower, tomato, squash, and tea,amongst others, or the seeds of any plant specifically named above or atissue, cell or organ culture of any of the above species The presentinvention is applicable to any plant, in particular a monocotyledonousplants and dicotyledonous plants including a fodder or forage legume,ornamental plant, food crop, tree, or shrub selected from the listcomprising Acacia spp., Acer. spp., Actinidia spp., Aesculus spp.,Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp.,Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaeaplurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkeaafricana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camelliasinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens,Chaenomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermummopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumisspp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeriajaponica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergiamonetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa,Diheteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum,Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Eragrestisspp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulaliavillosa, Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flemingiaspp, Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtiacoleosperma, Hedysarum spp., Hemarthia altissima, Heteropogon contortus,Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypertheliadissoluta, Indigo incamata, Iris spp., Leptarrhena pyrolifolia,Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex,Lotonus bainesai, Lotus spp., Macrotyloma axillare, Malus spp., Manihotesculenta, Medicago sativa, Metasequoia glyptostroboides, Musasapientum, Nicotianum spp., Onobrychis spp., Omithopus spp., Oryza spp.,Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp.,Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp.,Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthriafleckii, Pogonarthria squarrosa, Populus spp., Prosopis cineraria,Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercusspp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis,Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubusspp., Salix spp., Schyzachynum sanguineum, Sciadopitys verticillata,Sequoia sempemrens, Sequoiadendron giganteum, Sorghum bicolor, Spinaciaspp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthoshumilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifoliumspp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp.Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays,amaranth, artichoke, asparagus, broccoli, brussel sprout, cabbage,canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil,oilseed rape, okra, onion, potato, rice, soybean, straw, sugarbeet,sugar cane, sunflower, tomato, squash, and tea, amongst others, or theseeds of any plant specifically named above or a tissue, cell or organculture of any of the above species

“Cereal” comprises crop plants with edible grain for example plantsbelonging to the grass family that is cultivated for its nutritiousgrains such as oats, barley, rye, wheat, rice, and corn etc.

Within the scope of the present invention is also the application of theTwo-hybrid system, wherein any of the sequences of the present inventionare used to study interactions with other factors such as proteins. With“yeast two-hybrid assay” is meant an assay that is based on theobservation that many eukaryotic transcription factors comprise twodomains, a DNA-binding domain (DB) and an activation domain (AD) which,when physically separated (i.e. disruption of the covalent linkage) donot effectuate target gene expression. Two proteins able to interactphysically with one of said proteins fused to DB and the other of saidproteins fused to AD will re-unite the DB and AD domains of thetranscription factor resulting in target gene expression. The targetgene in the yeast two-hybrid assay is usually a reporter gene such asthe β-galactosidase gene. Interaction between protein partners in theyeast two-hybrid assay can thus be quantified by measuring the activityof the reporter gene product (Bartel & Fields 1997). Alternatively, amammalian two-hybrid system can be used which includes e.g. a chimericgreen fluorescent protein encoding reporter gene (Shioda et al. 2000).Yet another alternative consists of a bacterial two-hybrid system usinge.g. HIS as reporter gene (Joung et al. 2000). A person skilled in theart will also recognise that in adapted versions of the two-hybridsystem and also other techniques (e.g. gel-retardation,immunoprecipitation, competitive inhibition), it is possible to studyprotein-oligonucleotide interactions, and therefor these techniqueswhich use any of the sequences of the present invention are also withinthe scope of the invention.

The term “fragment of a sequence” or “part of a sequence” means atruncated sequence of the original sequence referred to. The truncatedsequence (nucleic acid or protein sequence) can vary widely in length;the minimum size being a sequence of sufficient size to provide asequence with at least a comparable function and/or activity or theoriginal sequence referred to, while the maximum size is not critical.In some applications, the maximum size usually is not substantiallygreater than that required to provide the desired activity and/orfunction(s) of the original sequence. Typically, the truncated aminoacid or nucleotide sequence will range from about 5 to about 60 aminoacids in length. More typically, however, the sequence will be a maximumof about 50 amino acids in length, preferably a maximum of about 60amino acids. It is usually desirable to select sequences of at leastabout 10, 12 or 15 amino acids or nucleotides, up to a maximum of about20 or 25 amino acids or nucleotides.

The invention also includes methods for high throughput compoundscreening. The man skilled in the art can easily design such methodsusing one or several elements of the invention.

The compounds yet to be obtained or identified can be compounds that areable to bind to any of the nucleic acids, peptides or proteins involvedin the process of processing precursor messenger RNA and are thereforeuseful in the method of the present invention. Said compound orplurality of compounds may be comprised in, for example, samples, e.g.,cell extracts from, e.g., plants, animals or micro-organisms.Furthermore, said compound(s) may be known in the art but hitherto notknown to be capable of suppressing or activating proteins involved inprocessing of precursor messenger RNA.

In the scope of the present invention is also included the introductioninto a plant cell one or more recombinant nucleic acid molecules, suchas a DNA molecule encoding a protein which when expressed in said plantcell at an effective amount increases or induces the expression of anendogenous polynucleotide acid according to the present invention or asdefined in claims or increases or induces the activity of a polypeptideof claim.

The present invention is further described by reference to the followingnon-limiting figures and examples.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1

Salt-tolerance phenotypes of yeast strains expressing the isolatedArabidopsis cDNA clones, U1A, Ct-SRL1 or RCY1, as determined by“drop-tests”; in which serial dilutions of saturated cultures of thedifferent strains, and of the control strain containing the emptyexpression vector, are tested on plates, without salt or containing theindicated LiCl or NaCl concentrations.

FIG. 2

Amino acid sequences derived from the nucleotide sequences of clonesRCY1 (A: SEQ ID NO 4) and Ct-SRL1 (B: SEQ ID NO 3). Dipeptides Arg-Ser.Arg-Glu and Arg-Asp. which define the RS domain, are written in boldletters. The underlined M in (A) marks the methionine residue used asinitiator during expression in yeast of the RS domain of RCY1 (SEQ ID NO21).

FIG. 3

Salt-tolerance phenotype of yeast strains expressing the amino-terminal(cyclin) domain of RCY1, its carboxy-terminal RS domain, or thefull-length protein. Drop-tests were performed as in the experiment ofFIG. 1.

FIG. 4

Salt-tolerance phenotype of transgenic Arabidopsis plants. T2 seeds fromthree independent transgenic lines (L1, L3 and L5, as an example of the12 obtained lines), transformed with the Ct-SRL 1 cDNA under control ofthe 35S promoter from CaMV, were germinated on agar plates without (A)or with (B) 20mM LiCl. Seeds from wild-type plants (Wt) were used ascontrol. All transgenic lines showed similar phenotypes.

FIG. 5

Sequences of the genes and proteins of the present invention.

EXAMPLES Example 1 Plant Material

Seeds of the red beet (Beta vulgaris var. DITA, also referred to hereinas “sugar beet”), were sown on pots containing a mixture of sand andvermiculite (1:1 w/w). The plants were grown under greenhouse conditions(8 hours at 20° C., 16 hours at 25° C. with supplementary lighting tostimulate a minimum of 12 hours photoperiod). They were periodicallyirrigated with a nutrient solution containing 2.4 g/l Ca(NO₃)₂.4H₂O, 1g/l KNO₃, 1 g/l MgSO₄.7H₂O, 0.3 g/l KH₂PO₄, 5.6 mg/l Fe-quelate(Kelantren, Bayer), 1.1 mg/l ZnSO₄.7H₂O, 3.3 mg/l MnO₄.H₂O, 0.3 mg/lCuSO₄.5H₂O, 3.8 mg/l H₃BO₃, 0.18 mg/l (NH₄)6Mo₇.4H₂O. For theconstruction of the cDNA library, three-week-old plants were irrigatedwith 200 mM NaCl for 24 hours before harvesting.

Example 2 Yeast Strains and Culture Conditions

The Saccharomyces cerevisiae competent cells W303-1A (MATa ura3, leu2,his3, trp1, ade2, ena 1-4::HIS3) were transformed with the ArabidopsiscDNA library.

The Saccharomyces cerevisiae strain JM26 (MATa leu 2-3,112 ura 3-1trp1-1, ade 2-1 his3-11,15 can 1-100, ena 1-4::HIS3, nha1:TRP1) providedby J. M. Mulet (Universidad Politécnica de Valencia, Instituto deBiologia Molecular y Cellular de Plantas) was used for the screening ofthe red beet cDNA library. Strain JM26 is a derivative of W303.1 A(Wallis et al., 1989, Cell 58: 409-419) with null mutations of the genesENA1-4 and NHA1, encoding a Na⁺-pumping ATPase and a Na⁺/H⁺antiporter,respectively, responsible for most of the yeast sodium extrusion(Garciadeblas et al. 1993, Mol. Gen. Genet. 236, 363-368), (Bañuelos etal. 1998, Microbiology 144: 2749-2758).

The yeast cells were grown in either minimal synthetic glucose medium(SD) or rich medium (YPD). SD medium contained 2% glucose, 0.7% yeastnitrogen base without amino acids and 50 mM succinic acid, adjusted topH 5 with Tris, plus the required amino acids [100 μg/ml leucine, 30μg/ml adenine, 100 μg/ml methionine] as indicated. YPD medium contained1% yeast extract, 2% Bacto peptone and 2% glucose. Media weresupplemented with NaCl and LiCl as indicated in the figures and theexamples. Solid media contained 2% bacteriological-grade agar.

Example 3 Construction of the cDNA Libraries

The construction of the Arabidopsis thaliana cDNA library in the vectorpFL61 is described in Minet et al. (1992) Plant J. 2(3): 417-422.

For the construction of a red beet cDNA library induced by salt stress,the plant material as described in example 1 was used. Directional cDNAswere synthesised (cDNA synthesis kit, Stratagene) using poly(A)⁺ RNAprepared from leaves of salt-treated red beet plants. cDNAs were ligatedinto phage λPG15 vector and packaged using a Gigapack III gold packagingextract (Stratagene). This phage has inserted the excisable expressionplasmid pYPGE15 (URA3 as a selection marker) that is usable directly forboth E. coli and yeast complementation (Brunelli and Pall, 1993, Yeast9: 1309-1318). A plasmid cDNA library was recovered from λPG15 by thecre-lox recombinase system (Brunelli and Pall, 1993, Yeast 9:1309-1318).

Example 4 Screening and Isolation of cDNA Clones Conferring SaltTolerance to Yeast

To screen for Arabidopsis thaliana cDNAs which increase salt tolerancein yeast, the cDNA library constructed in pFL61 was used to transformthe yeast strain W303-1A by the LiCl method (Gietz et al. 1992, NucleicAcids Res. 20: 1425). Transformants were screened for halotolerance inplates with minimal medium plus 25 mM and 50 mM LiCl, or as indicated inthe figures, and containing 400 μM methionine. Resistant clones weresubjected to fluoroorotic acid-induced plasmid loss (Boeke et al (1984)Mol. Gen. Genet. 197: 354-346) to select only those clones showingplasmid dependent LiCl tolerance. Results were confirmed in wild typestrain and in the double mutant ena1-4::HIS3 tfp1::LEU2, defective inthe vacuolar transport.

To screen for red beet cDNAs which increase salt tolerance in yeast, thecDNA library constructed in pYPGE15 was used to transform the yeastmutant strain JM26. Transformants selected on SD plates with leucine andadenine by uracil prototrophy were pooled and replated on screeningmedium (SD with leucine, adenine and methionine supplemented with 0.15 MNaCl) at a density of 2×10⁵ cells per plate (12×12 cm). Methionine wasadded to the selective medium to avoid selection of the HAL2-likehomologues already found in Arabidopsis (Quintero et al. 1996, PlantCell 8: 529-537) (Gil-Mascarell et al. 1999, Plant J. 17(4): 373-383).Alternatively, for the selection of Li+resistant yeast cells, thetransformants were replated on screening medium (SD with leucine andadenin supplemented with 20 mM LiCl). The putative positive clones wererescreened on the same NaCl or LiCl medium.

Example 5 Determination of Intracellular Lithium Content

Yeast cells expressing the cDNA clones of the present invention or thecontrol strain transformed with the empty vector, were grown toexponential phase and the medium was then supplemented with LiCl to 30mM final concentration. 10 ml—samples were taken at different times andthe intracellular lithium content was determined as described previously(Murgùia et al. 1996, J. Biol. Chem. 271: 29029-29033).

Example 6 Beta Galactosidase Assay

Yeast cells containing the E. Coli LacZ gene, interrupted or not with anintron (Legrain and Rosbash (1989), cell, 75: 573-583), under control ofan galactose-inducible promoter, were transformed with the Ct-SRL1 cDNAin a yeast expression vector or, as a control, with the empty plasmid.Cultures were grown to exponential phase in glucose minimal medium, withor without 35 mM LCl, and then shifted to galactose medium, maintainingthe same salt conditions, to induce the beta-galactosidase expression.Samples were collected at 0 (background value) and 4 hours afterinduction, and beta-galactosidase activity was measured in permeabilisedcells as described (Serrano et al, 1973: Eur. J. Biochem. 34: 479-482).

Example 7 RT-PCR Assay

Yeast cells overexpressing the Ct-SRL cDNA or transformed with the emptyvector were grown to exponential phase; the medium was then supplementedwith LiCl to 150 mM final concentration and total RNA was purified atdifferent times. Equal amounts of total RNA were digested withRNase-free DNasel, and reverse-transcribed with M-MuLV RTase (RocheMolecular Biochemicals) using primer preSARlrp (5′ CATCAAATCTTTCAGGG-3′:SEQ ID NO. 23), that hybridises to the second SAR1 exon 277 nucleotidesdownstream from the 3′-splice site. It was followed by 15 cycles of PCRamplification with Netzyme (Molecular Netline Bioproducts) DNApolymerase, and primer preSAR1fp, (5′-CTTTATTTTACTGTACAG-3′: SEQ ID NO.24), which hybridises to the 3′ end of the SAR1 intron. [α-³²P]dCTP(740kBq, 110 TBq/mmol, Amersham) was added after the 5^(th) cycle andthe products were resolved in 6% PA-urea gels and visualised byautoradiography. The relative intensity of the amplified bands wasdetermined using a FUJI (Fujifilm BAS-1500) phosphorimager. Sampleslacking reverse transcriptase ware used as controls.

Example 8 Transformation of Plants

The clones of the present invention, for example the Arabidopsis Ct-SRL1cDNA, are subcloned into pBI121 (Clontech), in place of the GUS gene,and the resulting binary vector is introduced into the Agrobacteriumtumefaciens strain C58C1 by electroporation. Transgenic plants, likeArabidopsis plants were obtained by in vivo infiltration as described inwww.arabidopsis.org/protocols_Mundy2.html#trans.inf.

Alternatively, the genes of the present invention can be transformed toother dicotyledon or monocotyledon plants. Therefore they are cloned inthe suitable plant transformation vector, such as a binary vector, underthe control of plant operable regulatory sequences, such as a plantoperable promoter and a plant operable terminator. These vectorscomprising a gene of the present invention can be transformed intoplants (such as a crop plant) using standard techniques well known bythe person skilled in the art, such as Agrobacterium mediated genetransfer. The transgenic plant expressing the gene of the presentinvention are expected to show an increased tolerance to environmentalstress, such as for example an increased tolerance to mineral salttoxicity. This increased tolerance can for example be observed by theability of the transgenic plants to germinate in salt containing medium.

Example 9 Rice Transformation with the Genes of the Present Invention

The expression of red beet genes or Arabidopsis thaliana that areinvolved in salt tolerance in yeast in monocotyledons, such as forexample Rice, can confer stress tolerance to that monocotyledoneousplant.

To transfer the stress tolerance activity of the genes of the presentinvention to monocots, the aforementioned genes (SEQ ID NO. 1, 2, 5, 7,9, 11, 13, 15, 17 and 19), operably linked to a promoter, are eachtransformed to rice using the standard transformation procedures wellknown to the persons skilled in the art and outlined in the followingparagraph. After several time periods ranging from 1 day to 1 or moreweeks, the seedling is checked for the expression of the transformedgene. This is done by growing the seedlings in organogenesis medium, andchecking the presence of the DNA or mRNA by PCR or reverse PCR. Afterthe confirmation of gene expression the transformed rice plants arechecked for the enhanced tolerance to stress situations including salt,drought and cold (see WO97/13843). This is done by growing thetransformed rice plants in medium containing increased amounts of NaClor LiCl. Also the increased resistance to cold or drought is tested bygrowing the transformed plants in suboptimal growing temperatures andsuboptimal levels of humidity, respectively (WO97/13843).

Agrobacterium-Mediated Rice Transformation

The genes of the present invention are operably linked to a promoter andcloned into a vector. These vectors are transformed to Agrobacteriumtumefaciens strain LBA4404 or C58 by means of electroporation andsubsequently transformed bacterial cells are selected on a solid agarmedium containing the appropriate antibiotics.

For demonstration of the expression of the genes of the currentinvention in rice, 309 mature dry seeds of the rice japonica cultivarsNipponbare or Taipei are dehusked, sterilised and germinated on a mediumcontaining 2,4-dichlorophenoxyacetic acid (2,4-D). After incubation inthe dark for four weeks, embryogenic, scutellum-derived calli areexcised and propagated on the same medium. Selected embryogenic callusis then co-cultivated with Agrobacterium. Co-cultivated callus is grownon 2,4D-containing medium for 4 to 5 weeks in the dark in the presenceof a suitable concentration of the appropriate selective agent. Duringthis period, rapidly growing resistant callus islands develop. Aftertransfer of this material to a medium with a reduced concentration of2,4-D and incubation in the light, the embryogenic potential is releasedand shoots develop in the next four to five weeks. Shoots are excisedfrom the callus and incubated for one week on an auxin-containing mediumfrom which they can be transferred to the soil. Hardened shoots aregrown under high humidity and short days in a phytotron. Seeds can beharvested three to five months after transplanting. The method yieldssingle locus transformants at a rate of over 50% (Chan et al. 1993,Plant Mol. Biol. 22 :491-506) (Hiei et al. 1994, Plant J. 6: 271-282)

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1. A method of enhancing salt stress tolerance in plants, plant cells oryeast cells, said method comprising overexpression of a nucleic acidmolecule encoding an RS domain (SEQ ID NO:21) in said plants or saidcells.
 2. The method according to claim 1 which comprises overexpressionof a nucleic acid molecule encoding an SR-like protein comprising saidRS domain.
 3. The method according to claim 2 wherein said SR-likeprotein is RCY1 (SEQ ID NO: 4).
 4. The method according to claim 3wherein the nucleic acid molecule encoding the SR-like protein has thesequence of SEQ ID NO:
 2. 5. An isolated nucleic acid molecule encodinga protein selected from the group consisting of: (a) a nucleic acidmolecule comprising the DNA sequence of SEQ ID NO: 2 or the complementthereof, (b) a nucleic acid molecule comprising the corresponding RNAsequence of SEQ ID NO: 2 as in (a) or the complement thereof, (c) anucleic acid molecule encoding a protein having the amino acid sequenceof SEQ ID NO: 4 or 21 or as defined in (a) or (b) characterized in thatsaid sequence is DNA, cDNA, genomic DNA or synthetic DNA.
 6. A vectorcomprising a nucleic acid molecule according to claim
 5. 7. A vectoraccording to claim 6 which is an expression vector wherein the nucleicacid molecule is operably linked to one or more control sequencesallowing the expression of said nucleic acid molecule in prokaryoticand/or eukaryotic host cells.
 8. A host cell comprising a nucleic acidmolecule according to claim 5 or a vector according to claim 6 or
 7. 9.A host cell comprising the vector of claim 7 wherein the host cell is abacterial, insect, fungal, yeast, plant or animal cell.
 10. An isolatedpolypeptide encoded by at least one of the nucleic acid moleculesdefined in claim
 5. 11. The polypeptide of claim 10 comprising the aminoacid sequence of SEQ ID NO: 4 or
 21. 12. A method of producing apolypeptide according to claim 10 or 11 comprising culturing a host cellunder conditions allowing expression of the polypeptide wherein saidhost cell comprises a vector comprising a nucleic acid molecule operablylinked to one or more control sequences allowing expression of saidnucleic acid molecule in prokaryotic or eukaryotic host cells andwherein the nuclelic acid molecule is selected from the group consistingof (a) a nucleic acid molecule comprising the DNA sequence of SEQ ID NO:2 or the complement thereof, (b) a nucleic acid molecule comprising thecorresponding RNA sequence of SEQ ID NO: 2 as in (a) or the complementthereof, (c) a nucleic acid molecule encoding a protein having the aminoacid sequence of SEQ ID NO: 4 or 21 or as defined in (a) or (b)characterized in that said sequence is DNA, cDNA, genomic DNA orsynthetic DNA, and recovering the produced polypeptide from the culture.13. A method for the production of a transgenic plant, plant cell orplant tissue comprising the introduction of a nucleic acid moleculeaccording to claim 5 in an expressible format or a vector in said plant,plant cell or plant tissue.
 14. A method for effecting the expression ofa polypeptide of claim 10 or 11 comprising introduction and stableintegration into the genome of a plant cell, of a nucleic acid moleculeoperably linked to one or more control sequences or a vector comprisinga nucleic acid molecule operably linked to one or more controlsequences, said nucleic acid molecule selected from the group consistingof (a) a nucleic acid molecule comprising the DNA sequence of SEQ ID NO:2 or the complement thereof, (b) a nucleic acid molecule comprising thecorresponding RNA sequence of SEQ ID NO: 2 as in (a) or the complementthereof; and (c) a nucleic acid molecule encoding a protein having theamino acid sequence of SEQ ID NO: 4 or 21 or as defined in (a) or (b)characterized in that said sequence is DNA, cDNA, genomic DNA orsynthetic DNA.
 15. The method of claim 13 further comprisingregenerating a plant from said plant cell.
 16. The transgenic plant cellobtainable by a method of claim 15 wherein said nucleic acid molecule isstably integrated into the genome of said plant cell.
 17. A Transgenicplant tolerant to salt stress as a result of the expression of at leastone of the nucleic acid molecules of claim
 5. 18. A Transgenic plantwhich as a result of the expression of at least one of the nucleic acidmolecules of claim 5 shows an alteration of its phenotype.
 19. Aharvestable part of a plant of claim 16 wherein said harvestable partcomprises the nucleic acid molecule which was introduced into thetransgenic plant.
 20. The harvestable part of a plant of claim 19selected from the group consisting of seeds, leaves, fruits, stemcultures, rhizomes, roots, tubers and bulbs.
 21. Transgenic progenyderived from any of the plants or plant parts of claim 17 wherein saidtransgenic progeny comprises the nucleic acid molecule which wasintroduced into the parent plant.
 22. The method of claim 1 forincreasing yield of the harvestable biomass of plants.
 23. A diagnosticcomposition comprising a nucleic acid molecule of claim
 5. 24. Aharvestable part of a plant of claim 18 wherein said harvestable partcomprises the nucleic acid molecule which was introduced into thetransgenic plant.
 25. Transgenic progeny derived from any of the plantsor plant parts of claim 18 wherein said transgenic progeny comprises thenucleic acid molecule which was introduced into the parent plant. 26.The method according to any of claims 1, 2 or 3 wherein said plant isArabidopsis thaliana or said plant cells are Arabidopsis thaliana cells.27. The method according to any of claims 1, 2 or 3 wherein said yeastcells are Saccharomyces cerevisiae cells.
 28. A diagnostic compositioncomprising a vector of claim 6 or
 7. 29. A diagnostic compositioncomprising a polypeptide of claim 10 or 11.